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THESIS

MICHIGAN STATE

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This is to certify that the

dissertation entitled

UREASE METALLOCENTER ASSEMBLY IN
KLEBSIELLA AEROGENES

 

presented by

Mary Beth C. Moncrief

has been accepted towards fulfillment
of the requirements for

Ph . D . degree in Bimhfimism

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MSU Ie An Afflnnettve ActIorVEqual Opportunlty Inetltulon
Wm:

 

UREASE METALLOCENTER ASSEMBLY IN KLEBSIELLA AEROGENES

By

MARY BETH C. MONCRIEF

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1996

ABSTRACT

UREASE METALLOCENTER ASSEMBLY IN KLEBSIELLA AEROGENES

By

Mary Beth C. Moncrief

Urease is a nickel-containing enzyme that functions in nitrogen metabolism and
participates as a virulence factor of several human pathogens. The protein contains a
binuclear active site where the metal ions are bridged by a lysine carbamate.
Metallocenter assembly in Klebsiella aerogenes urease requires the participation of four
accessory proteins: UreD, UreE, UreF, and UreG. Previous studies suggested that UreE
fimctions as a nickel donor, but roles for the other components were unknown.

I demonstrated that urease apoprotein could be activated in vitro and, using
deletion derivatives, showed that UreD played a key role in this process. I overexpressed
ureF and studied the insoluble gene product. l identified a series of complexes containing
UreD, UreF, and urease apoprotein and characterized their properties. My results suggest
that UreF modulates the UreD-urease apoprotein complexes activation properties by
excluding nickel ions from the active site until after the carbamylated lysine metallocenter
ligand forms. I purified UreG and showed that, despite having a P-loop motif, it does not
bind or hydrolyze nucleotides. Site-directed mutagenesis of the motif resulted in no
detectable urease activity; thus the motif is essential in viva. I partially purified a complex

containing UreD, UreF, and UreG and showed that, unlike UreG, it bound to an ATP-linked

resin. I showed that the UreG P-loop was not involved in UreD-UreF—UreG complex
formation, but was needed for binding to an ATP-linked resin. Based on these studies, I
propose a model for urease metallocenter assembly.

In other studies, I characterized the activity of crystalline urease and further
examined the apoprotein. I demonstrated that the enzyme is essentially inactive in the
crystal lattice and I resolved purified urease apoprotein into a series of bands by
nondenaturing gel electrophoresis. I showed that these species are likely to arise, in part,

by mixed disulfide formations involving Cys-319.

T 0 my parents, John and Pat Carr, and my husband, Dan, with love.

iv

 

ACKNOWLEDGMENTS

I would like to thank Dr. Robert Hausinger for being my mentor and providing an
inexhaustable supply of guidance and support. I thank the members of my guidance
committee: Dr. Shelagh Ferguson-Miller, Dr. Paul Kindel, Dr. Lee Kroos, and Dr.
Martha Mulks for their direction, support, and advice. I appreciate the technical advice as
well as support from current and previous members of the lab (Tim Brayman, Gerry
Coplas, Debbie Hogan, Linda Michel, Taha Taha, and Ruth Wallace).

I would like to thank Dr. Pao-Chi Liao and Dr. Doug Gage for their work on the
MALDI-mass spectrometry of UreG, and Il-Seon Park for the Escherichia coli (pKAUG—
1) strain. A special thank you goes to Dr. Tom Deits whose lab I worked in during a
rotation session and who has provided encouragement and support when needed. My
appreciation also goes out to Dr. Evelyn Jabri and Dr. Andy Karplus for their
crystallographic studies on urease and for providing me with their crystallization protocol
that I used for the activation studies in Chapter 2.

Finally, I would like to thank my parents and my husband for all the support,

understanding, and patience that they gave me during the years.

TABLE OF CONTENTS

LIST OF TABLES .............................................................................................................. vi
LIST OF FIGURES ........................................................................................................... vii
CHAPTER 1
INTRODUCTION ................................................................................................................ 1
Urease significance and structure ............................................................................. 2
Urease metallocenter assembly ................................................................................ 7
Urease metallocenter stability and in vitro activation of
urease apoprotein ......................................................................................... 8
Identification of accessory genes required for bacterial
urease activation ......................................................................................... 10
Postulated role of UreD as a urease-specific chaperone protein ................ 13
Potential role of UreE as a nickel-donor .................................................... 17
Characterization of UreF ............................................................................ 20
Characterization of UreG ........................................................................... 23
Current model for bacterial urease metallocenter assembly ...................... 27
Accessory genes required for urease activation in eukaryotes ................... 31
Summary ................................................................................................................ 32
Thesis Outline ........................................................................................................ 33
References .............................................................................................................. 36
CHAPTER 2
UREASE ACTIVITY IN THE CRYSTALLINE STATE ................................................. 46
Abstract .................................................................................................................. 47
Introduction ............................................................................................................ 48
Materials and Methods ........................................................................................... 49
Enzyme Purification and crystallization .................................................... 49
Assays ........................................................................................................ 49
Salt inhibition of urease activity ................................................................ 50
Results and Discussion .......................................................................................... 50
Activity of crystalline urease ...................................................................... 50
Kinetic analysis of salt inhibition ............................................................... 54
References .............................................................................................................. 57

vi

CHAPTER 3
FIRST DEMONSTRATION OF IN VITRO ACTIVATION OF UREASE

APOPROTEIN ................................................................................................................... 58
Abstract .................................................................................................................. 59
Introduction ............................................................................................................ 60
Materials and Methods ........................................................................................... 61

Cell growth and disruption ......................................................................... 62

Urease activity assays ................................................................................. 62
Results and Discussion .......................................................................................... 62

In vitro activation of urease apoprotein ..................................................... 62
References ............................................................................................................. 67

CHAPTER 4

PURIFICATION AND ACTIVATION PROPERTIES OF URED-UREF-UREASE

APOPROTEIN COMPLEXES ............................................................................................... 70
Abstract ....................................................................................................................... 71
Introduction ................................................................................................................. 72
Materials and Methods ............................................................................................... 74

Bacterial strains and plasmid constructions ................................................... 74
Culture conditions and cell disruption ........................................................... 76
Purification of DF-Apo .................................................................................. 76
Polyacrylamide gel electrophoresis ................................................................ 77
Urease activity assays ..................................................................................... 78
Activation of urease apoproteins .................................................................... 78
Results and Discussion ............................................................................................... 78
Purification and characterization of UreD-UreF-urease apoprotein
complexes ....................................................................................................... 78
Activation properties of DF-Apo ................................................................... 84
Function of UreF ............................................................................................ 86
References ................................................................................................................... 92
CHAPTER 5

CHARACTERIZATION OF UREG, IDENTIFICATION OF A URED-UREF-
UREG COMPLEX, AND EVIDENCE THAT A FUNCTIONAL
NUCLEOTIDE-BINDIN G SITE IS REQUIRED FOR IN VIVO

METALLOCENTER ASSEMBLY OF UREASE ................................................................ 94
Abstract ....................................................................................................................... 95
Introduction ................................................................................................................. 97
Materials and Methods ............................................................................................... 98

Bacterial strains and plasmid construction .................................................... 98
Culture conditions ........................................................................................ 100
Purification of UreG ..................................................................................... 100
Partial purification of the DF G complex ..................................................... 101
Preparation of polyclonal antibodies against UreG ..................................... 102

vii

Polyacrylamide gel electrophoresis .......................................................... 103

Urease activity assays ............................................................................... 103
Equilibrium dialysis determination of nucleotide binding ....................... 104
Chromatographic measurement of nucleotide hydrolysis ........................ 104
Carbonic anhydrase activity ..................................................................... 105
Results .................................................................................................................. 105
UreG purification and characterization .................................................... 105
Site-directed mutagenesis of UreG .......................................................... 108
Partial purification of the DF G complex ................................................. 108
Discussion ............................................................................................................ 1 12
UreG purification and partial characterization ......................................... 112
The requirement of the P-loop motif for UreG function .......................... 113
The DFG complex .................................................................................... 1 14
References ............................................................................................................ l 16
CHAPTER 6
PURIFICATION OF UREF AND CHARACTERIZATION OF NON-NATIVE
URED-UREF-UREG—UREASE APOPROTEIN COMPLEXES .................................... 119
Abstract ................................................................................................................ 120
Introduction .......................................................................................................... 121
Materials and Methods ......................................................................................... 122
Bacterial strains and plasmid construction .................................................. 122
Culture conditions and cell disruption ......................................................... 123
Partial purification of UreF ...................................................................... 124
Enrichment of DFG-Apo ......................................................................... 125
Polyacrylamide gel electrophoresis .......................................................... 126
Urease activity assays ............................................................................... 126
Activation of urease apoprotein ............................................................... 127
Results and Discussion ........................................................................................ 127
High level synthesis and partial purification of UreF .............................. 127
Enrichment of DFG-Apo ......................................................................... 132
References ............................................................................................................ l 3 7
CHAPTER 7
ADDITIONAL STUDIES ................................................................................................ 139
Identification of a series of urease apoprotein forms ........................................... 140
Incubation of Apo and UreD-UreF-UreG complex ............................................. 145
References ............................................................................................................ 149
CHAPTER 8
CONCLUSIONS AND FUTURE PROSPECTS ............................................................ 150
References ............................................................................................................ 160
APPENDD( I .................................................................................................................... 161

viii

CH!

LIST OF TABLES

CHAPTER 2
Table l-Specific activities of soluble and crystalline
K. aerogenes urease ........................................................................... 51

ix

 

CH4

CH3.

LIST OF FIGURES

CHAPTER 1
Figure l-Model of the urease bi—nickel active site ................................................... 5
Figure 2-The K. aerogenes urease gene cluster ..................................................... 12
Figure 3-Sequence comparison of selected UreD sequences ................................. 15
Figure 4-Sequence comparison of selected UreE sequences ................................. 18
Figure S-Sequence comparison of selected UreF sequences ................................. 22
Figure 6-Sequence comparison of selected UreG sequences ................................. 25
Figure 7-Model for urease metallocenter assembly ............................................... 30
CHAPTER 2
Figure l-Urease activity as a function of salt concentration .................................. 55
Figure 2-Kinetic analysis of U280; inhibition of urease ....................................... 56
CHAPTER 3
Figure 1-In vitro activation kinetics of urease apoprotein ..................................... 64
Figure 2-Urease stability during incubation at 37 oC ............................................. 66
CHAPTER 4
Figure l-Schematic representations of several plasmids
utilized in these studies ........................................................................... 75
Figure 2-Partial purification of the DF-Apo as analyzed by
SDS/polyacrylamide gel electrophoresis ................................................ 80
Figure 3-Native polyacrylamide gel electrophoretic comparison of the D-Apo
and DF-Apo samples ............................................................................... 82
Figure 4-Nickel ion concentration dependence of activation for the DF-Apo ....... 85
Figure S-Bicarbonate ion concentration dependence of activation for the
DF-Apo ........................................................................................................ 88
Figure 6-Effect of incubation of DF-Apo with metal ions prior to activation
by MC]; and bicarbonate ............................................................................. 90
Figure 7-Eflect of zinc ion concentration on DF-Apo activation .............................. 91
CHAPTER 5

Figure l-Purification of UreG as monitored by SDS/polyacrylamide

 

CHXP'

 

CH.—\P

C HAP

gel electrophoresis ................................................................................. 107
Figure 2-Effects of alterations to the UreG P-loop on formation of urease

apoprotein complexes ........................................................................... 109
Figure 3-Analysis of the UreD-UreF-UreG complex bound to an ATP-

linked agarose resin as analyzed by SDS/polyacrylamide

CHAPTER 6

CHAPTER 7

CHAPTER 8

 

gel electrophoresis ................................................................................. 1 10
Figure l-SDS/polyacrylamide gel electrophoretic analysis of

UreF solubilization ................................................................................. 129
Figure 2-Purification of ThioHisF as analyzed by SDS/polyacrylamide

gel electrophoresis ................................................................................. 130
Figure 3-Analysis of DFG-Apo complex purified from E. coli(pKAUD2F+)

by SDS/polyacrylamide gel electrophoresis .......................................... 133
Figure 4-Bicarbonate ion concentration dependence of activation

for the DFG-Apo* ................................................................................. 135
Figure S-Nickel ion concentration dependence of activation for

the DFG-Apo* ...................................................................................... 136
Figure l-Nondenaturing polyacrylamide gel electrophoretic

analysis of Apo ...................................................................................... 141
Figure 2-Nondenaturing polyacrylamide gel electrophoretic analysis of

purified Apo from E. coli(pKAU17C319AAureD) .............................. 144
Figure 3-SDS/polyacrylamide electrophoretic analysis of Apo and DFG

pool afier Superose 12 chromatography ............................................... 146
Figure 4-Native polyacrylamide gel electrophoretic analysis of Apo and DF G

pool afier Superose 12 chromatography ............................................... 147
Figure l-Proposed nucleotide hydrolysis scheme for the DF G complex ............. 157
Figure 2-Model for urease metallocenter assembly ............................................. 159

Apo
D-Apo
DF-Apo
DFG
DFG-Apo

DFG-Apo*

ABBREVIATIONS

urease apoprotein

UreD-urease apoprotein
UreD-UreF-urease apoprotein
UreD-UreF-UreG
UreD-UreF-UreG-urease apoprotein

UreD-UreF-UreG-urease apoprotein produced in
Escherichia coli(pKAUD2F+) cells

xii

CHAPTER]

INTRODUCTION

This chapter represents an updated version of a review chapter that was published as
Moncrief, M. B. C., and R. P. Hausinger (1996). Nickel incorporation into urease. In
Mechanisms of Metallocenter Assembly (R. P. Hausinger, G. L. Eichhom, and L. G.
Marzilli, eds.) VCH Publishers, Inc., New York, pp. 151-171 . I include a section that

outlines the remainder of the thesis.

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I. Urease Significance and Structure

Urease (EC 3.5.1.5) is a nickel-containing enzyme (1-3, 95) that catalyzes the
hydrolysis of urea to give one molecule each of ammonia and carbamate. Carbamate is
an unstable molecule and subsequently degrades to form carbonic acid and a second

molecule of ammonia:

H2N-CO-NH2 + H20 -) NH3+ HzN-COOH

HzN-COOH + H20 -) NH3 + H2CO3

This activity has been found in a large number of bacterial species, several fungi, selected
invertebrates, and a variety of plants. Urease from the jack bean plant has played an
important historical role in the field of biochemistry (1): It was the first enzyme ever to
be crystallized (4) as well as the first enzyme demonstrated to contain nickel (5).

The functional significance of urease differs for different organisms (reviewed in
reference 3). For example, in many soil microorganisms, the presence of this enzyme
allows cells to utilize urea as an environmentally important source of nitrogen (6). Soil
urea arises from mammalian excretion, metabolism of uric acid, or degradation of other
nitrogenous compounds. Similarly, urea is used as a nitrogen source by certain bacteria
that inhabit the rumen (7). In this case, a portion of the urea waste product generated by a
ruminant diffuses into the forestomach, urease activity of selected rumen microbes allows
for utilization of this nitrogen source, bacterial growth rates are enhanced, and the
bacteria ultimately serve as additional nutrient biomass for the animal. In contrast to
these situations where urease provides ammonia as a nutrient, the enzyme also functions
as a virulence factor in certain bacteria (3, 95). For example, urease activity has been

shown to be important in the development of urinary stones (8), acute pyelonephritis (9),

 

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urinary catheter obstruction (10), and peptic ulceration (l l, 12). In plants, the role of
urease is less clear, but it does appear to have an essential function. Because toxic
concentrations of urea accumulate in the leaves of plants grown in the absence of nickel
(13), a pathway of nitrogen flux involving urea as an intermediate has been proposed
(reviewed in reference 14). Recycling of urea-derived nitrogen by urease appears to be
especially important during seed germination (87). Polacco and Holland (14) have also
suggested that the embryo-specific urease of some plant seeds may play a role in chemical
defense against herbivores by causing hyperammonernia to occur, similar to that
generated by ureases of some pathogenic bacteria.

Urease metal contents and protein sequences are highly conserved among
eukaryotic and prokaryotic organisms (2). Jack bean urease is a homohexameric protein
containing two nickel ions per subunit (5). The primary sequence of the single
polypeptide (Mr 90,770; 840 amino acids) has been determined by both protein (15) and
DNA (16) sequencing methods. Similarly, atomic absorption analysis coupled with
inhibitor studies demonstrated the presence of two nickel ions per catalytic unit
(approximate Mr 100,000) in the enzyme isolated from Klebsiella aeragenes (17). In
contrast to the plant enzyme, however, protein characterization (18) and DNA sequence
analysis (19) of the genes encoding this bacterial enzyme indicates the presence of three
subunits (Mr 11,086, 11,695, and 60,304 for UreA, UreB, and UreC, respectively).
Furthermore, the size of the native bacterial protein is less than half that of the native jack
bean urease (18). Although difl‘ering in the number of distinct subunits and in quaternary
structure, the K. aerogenes and plant enzymes are greater than 50 percent identical in
protein sequence: UreA aligns with residues 1-101 of jack bean urease, UreB matches
residues 132-237, and UreC is homologous to residues 271-840.

The presence of multiple distinct subunits, each related in sequence to portions of
the single subunit jack bean enzyme, also has been observed (or inferred from DNA

sequence analysis) for ureases fi'om Proteus mirabilis (20), P. vulgaris (21), Ureaplasma

 

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urealyticum (22, 105), Helicobacter pylori (23, 24), H. felis (25), H. heilmannii (26), H.
mustelae (106), H. nemestrinae (107), Yersinia enterocolitica (27, 88), a thermophilic
Bacillus species (28), Rhizobium meliIoti (29), Staphylococcus xylosus (30), Haemophilus
influenzae (96), Bacillus pasteurii (97), Streptococcus salivarius (99), Mycobacterium
tuberculosis (100), and Lactobacillus fermentum (unpublished sequence submitted to
GenBank with accession number D10605). In most cases, genes for three subunits were
shown to be present, however, the five Helicobacter species possess genes that encode
only two urease subunits, where the smaller (~Mr 30,000) includes sequences similar to
those found in both of the smaller subunits of three-subunit enzymes. These results are
consistent with gene fusion or disruption events during evolution of this enzyme.
Although exceptions have been noted (31-33), the metal content of most bacterial ureases
that have been examined resembles that of the K. aerogenes enzyme [i.e., approximately
two nickel ions per large subunit (reviewed in reference 2)]. The high degree of sequence
similarity and the near identity in metal content observed among ureases is consistent
with the presence of the same general active site in each of these proteins.

The structure of the urease active site and the detailed mechanism of action for
this enzyme are best understood for the protein isolated from K. aerogenes. In particular,
the K. aerogenes enzyme has been crystallized (49) and the structure determined to 2.2 A
resolution and refined to an R factor of 18.2 percent (89). The enzyme is a tightly packed
trimer of trimers [(0437);] containing three bi-nickel active sites. Key features of the
catalytic center are illustrated in Figure 1. The two nickel ions are located 3.5 A apart
and are bridged by a carbamate formed on Lys-217. A solvent molecule is tightly bound
to one metal ion and appears to partially bridge to the second. One nickel ion possesses a
distorted trigonal bipyramidyl or distorted square pyrarnidyl geometry and the five ligands
include His-134, His-136, Asp-360, Lys-217 carbamate, and water (or hydroxide). The
second nickel ion possesses pseudotetrahedral geometry and is liganded by His-246, His-

272, Lys-2 l 7 carbamate, and solvent in partial occupancy.

 

Figure 1. Model of the urease bi-nickel active site. The enzyme possesses a novel
binickel center in which one nickel ion is coordinated to His-246, His-272, one oxygen
atom of a lysine carbamate, and, with partial occupancy, a solvent molecule (K
aerogenes numbering). The second nickel ion is coordinated by His-134, His-136, Asp-
360, the other oxygen atom of carbamylated Lys-217, and the solvent molecule (89).
Urea is thought to coordinate to the nickel ion on the left. The metal ion acts as a Lewis
acid, and the O-coordinated resonance form is proposed to be stabilized by interaction
with His-219 and one of several carboxylate groups at the active site. The water bound to
the second nickel ion, probably in its deprotonated form, carries out a nucleophilic attack
on the urea carbon atom to form a tetrahedral intermediate. Ammonia is released by a
general acid-assisted reaction (perhaps involving His-320), and, finally, carbamate is
released.

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In addition to the metallocenter ligands, the crystal structure reveals several
additional residues worth noting. His-219 is appropriately positioned to stabilize
substrate binding to the fourth ligand position of the second nickel ion. His-320 is
located near the bi-nickel center and is likely to participate in catalysis. Finally, Cys-219
is within the active site environment, but does not play a catalytic role.

Catalysis has been suggested to proceed by urea binding in O-coordination to the
fourth coordination site of the second nickel ion with stabilization rendered by His-219.
The solvent molecule that is now solely coordinated to the first nickel ion is thought to
carry out nucleophilic attack on the bound urea to form a tetrahedral intermediate.
Elimination of ammonia from the intermediate is facilitated by proton donation, possibly
involving His-320, and carbamate is released. This working model of urease catalysis is a
slight modification of that originally proposed by Zemer and colleagues for the jack bean
enzyme (43). The novel binuclear nickel-dependent active site of urease has been shown
to enhance the degradation of urea by at least 1014-fold over the uncatalyzed reaction
(43). Additional studies involving K. aerogenes urease are discussed below in terms of
the crystallographically determined structure and this mechanistic model. The binuclear
center of the K aerogenes enzyme has been probed by several spectroscopic and
biophysical methods. On the basis of X-ray absorption spectroscopic analyses, the two
nickel ions were thought to possess 5-coordinate geometry with approximately two
imidazoles bound per nickel and the remainder of the ligands being oxygen or nitrogen
donors (no thiols) (34). The crystal structure confirms that this is the geometry for one
nickel ion, but is an overestimate for the second nickel ion (89). Saturation
magnetization measurements provide no evidence for magnetic coupling of the two nickel
ions in the native enzyme (35), but rather are consistent with a pH-dependent equilibrium
between S=1 and S=0 metal ions. In the presence of a thiol, the metallocenter becomes
diamagnetic and clear evidence for antiferromagnetic coupling of the two nickel ions was

obtained by using variable temperature magnetic circular dichroism spectroscopy

 

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(Michael Johnson, personal communication), as previously reported for the jack bean
enzyme (36). Furthermore, X-ray absorption spectroscopic analysis revealed that the
apparent five-coordinate geometry is retained in thiol-inhibited enzyme and provided
evidence for the presence of Ni-S and Ni-Ni scattering components at 2.23 A and 3.26 A,
respectively (34).

Analysis of the metallocenter ligands has been examined by other methods. Site-
directed mutagenesis experiments were carried out to convert independently three highly
conserved histidine residues to alanine resulting in the loss of one (H134A) or both
(I-Il36A and H246A) of the nickel ions from the active site of the enzyme (37). An
additional urease metallocenter ligand has been suggested to arise from reaction of an
amino acid side-chain with a molecule of carbon dioxide (38). Based on precedent
involving ribulose 1,5-bisphosphate carboxylase/oxygenase, a reasonable possibility for
the identity of this ligand is a lysine carbamate. The crystal structure subsequently
confirmed that His-134, His-136, His-246, and the carbamate of Lys-217 were ligands.

Additional studies have been used to tentatively identify other residues at the

urease active site. An ionizable residue with pKa = 6.55 is required for catalysis (18) and

has been tentatively identified as His-320 from chemical modification studies and site-
directed mutagenesis methods (37, 39). Another ionizable group with pKa = 8.55 was
shown to participate in catalysis (l 8); this group was tentatively assigned to a Cys-319
interacting with another unidentified residue (40). Chemical derivatization of Cys-319
eliminates urease activity (41); however, C319A, C3198, and C319D mutant proteins
retain significant levels of activity and Cys-3 19 is now thought to play no catalytic role
despite its location at the active site (42). Finally, the presence of an acidic group at the
active site has been suggested on the basis of apparent electrostatic repulsion of
negatively charged inhibitors (17) and chemical reagents (41).

II. UREASE METALLOCENTER ASSEMBLY

 

 

 

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8

In vivo assembly of the urease metallocenter is a complex process that appears to
require energy and the participation of several auxiliary protein components. My
discussion of this topic begins by describing the stability of the urease metallocenter and
the requirements for its in vitro assembly. Evidence will then be presented to
demonstrate the presence of multiple accessory genes that are required for in viva
activation of bacterial urease. I will then detail known properties of the products encoded
by fan of these genes. By combining knowledge of the in vitro urease activation
requirements with my understanding of the accessory protein properties gleaned from
genetic and biochemical characterization studies, I will propose a model for the in vivo
activation mechanism of urease. Finally, I summarize evidence consistent with the

presence of similar accessory genes in eukaryotes.

A. Urease Metallocenter Stability and in vitro Activation of Urease Apoprotein

The nickel ions of urease are not removed by dialysis of the enzyme in buffers
containing 1 mM EDTA at neutral pH (1 , 42); however, harsh treatments with
denaturants or acid conditions result in an irreversible loss of nickel and urease activity
(44, 45). This evidence is consistent with the nickel metallocenter being buried in the
protein. Assembly of the urease metallocenter must then require either conformational
flexibility of the apoprotein to allow the nickel ions to penetrate into the metal binding

site or the assistance of additional cellular factors to allow partial unfolding of the

apoproteit
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apoprotein. As described later, in vitro and in vivo activation studies have been used to
address this question.

In vitro activation of urease had not been achieved until 1994. Urease apoproteins
(termed Apo) that are present in crude bacterial or plant cell extracts, obtained from
cultures grown in nickel-depleted media, generally have been found to be incapable of
activation by simple nickel ion addition or by providing nickel ion in the presence of
denaturants or stabilizing compounds (46-48). Partial in vitro urease activation was
achieved, however, by using freshly prepared, highly concentrated cell extracts from
nickel-depleted E. coli cultures that contain and overexpress plasmid-bome K. aerogenes
urease genes (48, Chapter 3). In this case, the activation process was both very slow,
reaching maximal specific activity after approximately 1 day, and far fiom complete, with
functional metal ion incorporation into only about 10 percent of the Apo that is present.
More recently, in vitro urease activation was realized using purified K aerogenes
apoprotein (3 8). The observed activation was complete in approximately three hours, and
it accounted for metallocenter assembly into nearly 30 percent of the total urease catalytic
sites. It was remarkable at the time that the in vitro process was shown to require carbon
dioxide in addition to nickel ion. The kinetics of activation were consistent with initial
interaction between carbon dioxide and the protein, followed by nickel binding. The
proposal put forward to explain this result was that an active site residue may react with
carbon dioxide to form a carbamate that subsequently serves as a metal-binding ligand,

much like the lysine that forms a carbamate involved in magnesium binding in ribulose

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ofprote.
ions
dieyclo:
urease _

accesst».

B. 1an

 

10
1,5-bisphosphate carboxylase/oxygenase (50). This hypothesis has been confirmed by the

crystal structure of the enzyme (89).

Although conditions now have been identified to at least partially activate purified
Apo, it is important to emphasize that urease activation in vitro is distinct in many ways
from that occurring in viva. One key distinguishing feature of the in vivo process is an
apparent energy dependence for urease activation, as exemplified by studies reported by
Lee et al. (46). Whereas preformed Apo undergoes slow activation starting 30 min after
nickel ion addition to nickel-depleted K. aerogenes cells that are treated with an inhibitor
of protein synthesis, the same cells fail to generate urease activity when exposed to nickel
ions following treatment with dinitrophenol (a protonophore) or
dicyclohexylcarbodiimide (an ATPase inhibitor). Moreover, as described below, in vivo
urease activation requires the participation of several auxiliary proteins encoded by

accessory genes.

B. Identification of Accessory Genes Required for Bacterial Urease Activation

Genetic analyses (e.g. deletion and transposon insertional mutagenesis) in a
variety of microorganisms have provided compelling evidence that additional genes,
other than those encoding the urease subunits, are required for urease activity (e.g., 28,
51-57). DNA sequencing efforts have identified several of these urease-related genes. In
K aerogenes, for example, seven genes make up the urease gene cluster (19, 57;

illustrated in Figure 2). Three of the genes (ureA, ureB, and ureC) encode the structural

II “('56

microbe

are

er

ll

subunits, whereas, an additional gene (ureD) is located immediately upstream of these
structural genes and three genes (ureE, ureF, and ureG) are located downstream.
Homologous urease accessory genes have been characterized for several other bacteria
[e.g., P. mirabilis (20, 58), a thermophilic Bacillus species (28), H. pylori (55), E. coli
(56), Y. enterocolitica (88), H. influenzae (96), B. pasteurii (97), S. salivarius (99), and
M. tuberculosis (100)]. The urease gene organization is well conserved in these
microbes, except that the ureD gene is positioned downstream of ureG in Bacillus TB-90 .
(28), Y. enterocolitica (88), S. salivarius (99), U. urealyticum (105), H. pylori (55), and in
H. influenzae (96), (in the latter two cases the gene is designated well). In addition to the
genes encoding the urease subunits and four accessory proteins, other genes are
sometimes present. For example, apparent regulatory genes have been identified in H.
pylori [termed ureC and ureD (24)], and in P. mirabilis [termed ureR (59)]. Furthermore,
a gene termed ureH was identified downstream of the four urease accessory genes of
Bacillus TB-90 (28). The predicted sequence of UreH is 23 percent identical to HoxN, a
high-afiinity nickel transport protein in Alcaligenes eutrophus (60). Another related
nickel-transport peptide, NixA, has been partially characterized from H. pylori where it
appears to be necessary for expression of active urease when grown in media possessing
low concentrations of nickel ion (90). The putative regulatory and nickel transport genes
in these microbes are not required for urease activation, whereas the ureD, ureF, and

ureG genes are all essential for generating active urease (24, 28, 57, 59).

A major advance in understanding the role of the four urease accessory genes in
enzyme activation was reported by Lee et al. (5 7) working with a plasmid-bome K

aerogenes urease gene cluster expressed in E. coli. These investigators studied the effects

1.1

 

301

Figure
Strum;

that ar

12

 

 

 

 

 

 

 

 

 

 

 

 

Urease subunits 1 kb
11.1 kDa 60.3 kDa
11.17 kDa
D A B C E F G
l ,
25.2 kDa

Urease accessory proteins

Figure 2. The K aerogenes urease gene cluster (19, 57). The urease gene cluster in K.
aerogenes is composed of seven genes. Three genes (ureA, ureB, and ureC) encode
structural subunits, and four genes (ureD, ureE, ureF, and ureG) encode accessory genes

that are required for functional assembly of the urease metallocenter.

   
  
 
   
 
   

of deletit
actitity
affect u
absence
subunit

corre

deletion

exthibit

sequent

Protein

13

of deletion mutants in each of the accessory genes on urease gene expression, enzymatic
activity and nickel content. They found that deletions in ureD, ureF, and ureG do not
affect urease subunit synthesis but result in the total loss of urease activity due to an
absence of nickel in the protein. Deletions in the ureE gene also have no effect on
subunit urease gene expression, but lead to a partial reduction in activity with a
corresponding decrease in nickel content. Co-transformation of E. coli cells with the
deletion derivatives and appropriate subclones of the missing genes located on
compatible plasmids demonstrated that the genes act via trans-acting factors. The
simplest interpretation of these results is that the four genes encode proteins that are
required for the functional incorporation of nickel into the active site of urease (57). In
the following sections 1 will detail the properties and speculate on possible roles for the

UreD, UreE, UreF, and UreG accessory proteins.

C. Postulated Role of UreD as a Urease-Specific Chaperone Protein

On the basis of ureD DNA sequencing results, UreD proteins are predicted to
exhibit limited amino acid sequence conservation (Figure 3) and they appear to be
unrelated to other sequences available in GenBank or other available data bases. The
sequences contain no obvious motifs that would provide insight into the function of this

protein in urease activation.

The only efl'ort to characterize the UreD protein is that reported for the product of
the K aerogenes ureD gene (48). In this microbe, ureD is expressed at very low levels

compared to the other urease genes (5 7) making the pmification and characterization of

14

Figure 3. Sequence comparison of selected UreD sequences. Sequence analysis was
performed using the Pileup program (61). The UreD sequences are from the following
species: kaured, K aerogenes (57, M55391); kpured, Klebsiella pneumonia
(unpublished, L07039); pmured, P. mirabilis (20, M31834); ecured, E. coli (94, L03307);
bacured, Bacillus sp. strain TB-90 (28, D14439); hpured, H. pylori (55, M84338); and
yeured, Y. enterocolitica (88, L24101).

 

a. 9. a. E a. a. a. .5. t. I. -

r. v. v. r. v. v. v. v. v. v. v. v. v. v. y. I ..

... H. .u .. .. .. 4 .u .4 ... .. .. a .4 .. J J . ._ .. . . .

2. C. I ~. 3 I. a. .2 C. .M A. .. C. .2 a I. T. C : C. a. .c C. 3. 3 C E I. .. .. . l.

I." . :. a. 2,. p” n... . a , . .y. .5 .3 a“ .... (a .. p... a. .: F“ ..v. .l.. I... r? a: 3. . .. .ru 3. e x: 3.. U: .r.. .K we. .2
: o~ kg .FL

 

kaured
kpured
pmured
ecured
bacured
hpured
yeured

kaured
kpured
pmured
ecured
bacured
hpured
yeured

kaured
kpured
pmured
ecured
bacured
hpured
yeured

kaured
kpured
pmured
ecured
bacured
hpured
yeured

kaured
kpured
pmured
ecured
bacured
hpured
yeured

kaured
kpured
pmured
ecured
bacured
hpured
yeured

kaured
kpured
pmured
ecured
bacured
hpured
yeured

MSEPEYRGNS

51

HQA...GGKT
QQA...GGKT
ELK...RGKT
ELK...RGVT
AKK...RQKT
KTKIGADGRC
AKR...EHRS

101

GDELTISAHL
GDELTISAHL
GDTLSININV
GDKLLINIDV
GDVYLTNLDV
GDAQDVQLNI
GDRLATDVIV

151

LPQDAIFFPG
LPQDAIFFPG
LPQENIFFPD
LPQENIFFPE
LLDPLISYKG
APFPLIPFEN
MPDPLIPHRN

201

LSNRLEVWV.
LSNRLEVWV.
VKGRLNFYV.
IRGRLQFYI.
IRSKLKVY..
LHTKISIL..
YSSRVAAHVF

251

LCYPATDALL
LCYPATDALL
YIYPATDALK
YIYPASDELK
LIFHQKADKT
VLVNCPIELS
ILLTPKEHHE

301

LICQRVMRDV
LICQRVMRDV
EPMMACFAQV
EPMMACEAHI
GIIENMISRA
EPLLHLREKI
AGVKNEIRQF

FTGSRHALGI

VLASAQHVGP
VLASAQHVGP
CLTEKRHLGP
RLTDKQHIGP
IISSCYHEGA
VIEDNFFTPP
ILAEMERRVP

APGCHTLITM
APGCHTLITM
QPYAHALLTT
QPHAHALLTT
EEEAELAVTT
GPNCKLRITS
EAGACAHVTT

ANARLFTTFH
ANARLFTTFH
AQVCLTTHIH
AQVRLETHVR
ARFIQETTVH
AHFKGNTTIS
SRFITDTTIN

...... DNEP
...... DDEP
...... DERL
...... DDKL
..... KKDRL
..... QDEKP
LGKEQPAGKE

DGVRDALA..
DGVRDALA..
EIIQHHLE..
AELHESLAVF
FLDRLYDEME
GVRGLIEESE
RILARVPAHF

WQFLRPHLTG
WQFLRPHLTG
WQIVRQHWLG
WQATRQYWLG
HSEARRELLG
ARFITQTITP
WKIAREEILG

15

NAPELAQYQD

LTVQRPFYPE
LTVQRPFYPE
LMVQRPFYPE
LMVQRPFYPE
LKVSRPIYLE
FKLMAPFYPK
SMVQKALYWD

PGASKFYRSS
PGASKFYRSS
PGATKFYRSA
PGATKFYRSA
QSATKVYKTP
QSFEKIHNTE
QSATKVHMMN

LCASSRLLAW
LCAFSRLLAW
LASSAKFIGW
IASSSKFISW
IEEDSGFFYS
LRSSSQLLYS
IHPTATAIYS

LLVERLHLQ.
LLVERLQLQ.
ILTESMRVE.
TLAESIFIE.
VLFDHLRLEP
IYYDNTILDP
LFVEKYVLEP

...... PLGL
...... PLGL
...KVNPLVE
FSTEVRPL.E
AFDSDVRFGM
GVDGAV....
DIKGGIASGA

KSPVLPRIWL
KSPVLPRIWL
YCPEPPRIWA
YCPEPPRIWA
KNGVTWRKY*
KV*. ......
VTLPEKFLWR

..VLPPLK.K
..VLPPLK.K
...MPDFSEK

..MSDFSGS

EPAQMRRRGV

EETCHL...Y
EETCHL...Y
QGVAHT...Y
QGIAHT...Y
KDLPFL...Y
DDLAEI...M
EEMPELPCVT

GAQALVRQQL
GAQALVRQQL
GGTASQTQTL
GGVARQVQTL
KKPVVQQTNI
DGFASRDMHI
ANYASQIQNF

DLLCLGRPVI
DLLCLGRPVI
EMQCFGRPVL
EIQCLGRPVL
DVITPGWA..
EIIVAGRV..
EVLMSGRKYH

..EGELSSIA
..EGELSSVA
..GLQKQAAA
..GSQKQSAV
D.EDMSGMLQ
KTTDLNNMCM
KSESLDAIGV

YAGASLTDRL
YAGASLTDRL
YGLTDV.DGI
YGLTDV.DGI
TSLP.ASG..
SEIA.SSH..
TRLPNDCG..

332

T*
T*
T*
T*

O O
i
O

50
GWQATLDLRF
GWQRTLDLRF
GWLADIALRY
GWLAEIFLRY
KCTGVLQLSA
TYAQESKLRL
GKSGYLKLRF

100
LLHPPGGIVG
LLHPPGGIVG
LLHPPGGVVG
LLHPPGGVVG
LIHVGGGYVD
LLAVSPGLMK
MISTSGCILQ

150
TLAPQATLEW
TLAPQATLEW
TVAQEGFLEW
TVAPNGFLEW
HLKKGSVLEY
VVGENAFLDF
TVEEGGYLEF

200
..GETFSHGT
..GETFSHGT
..NEWFETGK
..NEQFDNGD
EDGSLFPYDW
ARNELFKFNR
HADERFGFDV

250
ERPW..VGTL
ERPW..VGTL
MREFPMFGSL
MREFPMVGSL
MDGYTHIGTF
FDGYTHYLNL
MQSFDAFGNV

300
LTVRFLSDDN
LTVRFLSDDN
LVLRVLGTQT
LVLRLLGSQT
IILRILARST
LCLKALAKGS
LVFKALGIDS

 

 

 

 

tin

st:

51‘

ll!

16
the encoded protein very difficult. Speculating that the low expression of ureD may be

due to the presence of a poor ribosome binding site coupled with a GTG initiation codon,
site-directed mutagenesis was used to provide a more consensus-like ribosome binding
site and generate an ATG initiation codon. These changes led to the successful

overexpression of the ureD gene in E. coli. The UreD protein (Mr = 29,300) copurifies
with urease apoprotein, and several UreD-urease apoprotein complexes (termed D-Apo)

were shown to be formed. The results were rationalized in terms of the trimeric [(aBy)3]

Apo species binding 0, 1, 2, or 3 UreD molecules. When nickel is added to the partially
purified D-Apo complexes, rapid urease activation is observed with the process being
complete in approximately 60 minutes and the level of activity being directly correlated to
the number of UreD proteins that are associated with Apo. The complex containing 3
UreD molecules gives rise to the highest activity, but this maximal value represents only
around 30 percent of that expected for fully active urease protein. Coincident with the
activation of urease by added nickel, UreD dissociates fiom the enzyme. Based on cross-
linking studies of the D-Apo complexes, UreD appears to associate with UreB (the [3
submit) of the Apo species (Taha Taha, unpublished observations), and UreD was
proposed to act as a chaperone protein or molecular prop that stabilizes a urease
apoprotein conformation which is competent for nickel insertion (48). This role is
reminiscent of that proposed for MelCl in copper incorporation into tyrosinase (62, 63),
and for NifY in iron-molybdenum cofactor insertion into apodinitrogenase (64, 65). A

chaperone-type function for UreD remains a possibility, but the evidence is less

 

 

11L

C017.

des;

0.1

bar
Sir

Mr

M
his

to

l7

compelling with the recent demonstration of in vitro activation of purified Apo as

described in the previous section.

D. Potential Role of UreE as a Nickel-Donor

UreE proteins are also predicted to be conserved to only a limited extent in the
bacterial species for which ureE sequencing information is available (Figure 4).
Similarly, these proteins are not clearly related to other proteins in sequence data bases.
Most of the UreE sequences, however, do reveal several potential metal ion-binding sites.
In K aerogenes, for example, Cys-T‘yr-His and His-His-Asp-His sequences are found
within the protein and 10 of 15 residues at the carboxyl terminus are predicted to be
histidinyl residues (19). The first two regions are not conserved in any other UreE
proteins, but eight out of the last nine residues in the P. mirabilis protein (20) and 11 out
of the last 22 residues in Y. enterocolitica UreE (88) are histidines. This polyhistidine
motif was not, however, conserved in the H. pylori UreE sequence (24) and was limited
to only two histidines out of four residues at the Bacillus UreE tail (28). UreE sequences
from B. pasteurii (97), S. salivarius (99), and U. urealyticum (105) also lack a histidine-
rich tail, while H. influenza (96) contains this motif (sequence comparisons not shown).
It is possible that alternative proteins containing polyhistidine motifs can substitute for
the "defective" UreE molecules in these species. For example, several hypB sequences,
encoding proteins that participate in metallocenter assembly of the nickel-containing
hydrogenases, possess polyhistidine regions (e.g., 66-70). Furthermore, the nickel-
binding WI-IP protein fi'om E. coli was shown to possess a histidine-rich region (71).

The requirement for the histidine-rich tail region of the K aerogenes UreE protein
was tested by generation of a truncated protein lacking 15 residues at the carboxyl

terminus (102). The truncated UreE was nearly as effective as the wild-type UreE for

 

IV

In
D
n

In

 

kauree
pmuree
bacuree
hpuree
yeuree

kauree
pmuree
bacuree
hpuree
yeuree

kauree
pmuree
bacuree
hpuree
yeuree

kauree
pmuree
bacuree
hpuree
yeuree

kauree
pmuree
bacuree
hpuree
yeuree

kauree
pmuree
bacuree
hpuree
yeuree

0000000000

MGRPTMVGMR

51

AAATASVT..
STEKPKLT..
..... VPHIE
..... DFSVD
QEKLKDATFD

101

VLSNEEGTEF
ILLSEEG.DV
ILYMDDHNMI
ILFKEEKEII
VLAWDEKTNV

151

LQIMPGELRY
LQIEAGWCRY
AQFEG..N..
LYYGE..SQF
QHWKAVTKNN

201

AHHDHHAHSH
HHDHHH*...
8* ........
HSEPNFKVSL
NSEARLLFGG

DHSHSHGDHD

GIETDFITVG

...LPIDVRV
.LCLTMDERT
RVYMRSDDLV
YVDLEWFETR
LLVLDQREAQ

VQV ...... I
VTI ...... E
VI ....... 5
AV ....... N
AVVVQINLRD

HHDHVLDDML
FHDHVLDDMA
EMIVQYDYLV
EFKTPFEKPT
EVYVPLTVAT

.........
OOOOOOOOOO

HDHKH*

NWLLPKIQAR

KSRVKVTLND
KSRLKVALSD
KRVKRVVTDH
KKIARFKTRQ
KSRCRKLSTQ

AADEEVSVVR
AAKEQVSTVY
VLEDDVLTIK
ILDSEVIHIQ
VMVIDLSELK

RQFGLTVTFG
RGLGATVVVG
EELLQKLSIP
LALLEKLGVQ
TMMDSVMRTH

ASPLDEPHGS

...MLYLTQ.
...MKKFTQI
....MMVEKV
....MIIERL
SLYMILIEHI

GRDAGLLLPR
GQEAGLFLPR
GKBIGIRLKE
GKDIAVRLKD
GLDLGISLDR

CDDPFMLAKA
SDDPLLLARV
PTSMQQMGEI
AKSVAEVAKI
SRSPDELIKT

QLPFEPEAGA
LBKYQPEPGA
.FTRENRKMK
.NRVLSSKLD
GFQHLPFRFV

GLHVHAIHSH

50
..... RLEIP
IDQQKALELT
VGNITTLEKR
IGNLRDLNPL
LGNVKKDPVW

100
GLLL..RGGD
GTVL..KEGD
HQELQ..DGD
APKLGFSQGD
HVVLA..DGD

150
CYHLGNRHVP
CYHLGNRHVP
AHQLGNRHLP
CYEIGNRHAA
CFELGHALGN

200
YASESHGHHH
YGGSSGGHHH
QAFRPIGHRH
SKERLTVSMP
KGAEILPLLS

GTGHTHSHDH

The UreE sequences,
kauree, K

Figure 4. Sequence comparison of selected UreE sequences.
analyzed using the Pileup program (61) are from the following species:
aerogenes (19, M36068); pmuree, P. mirabilis (20, M31834); bacuree, Bacillus sp. strain
TB-90 (28, D14439); hpuree, H. pylori (55, M84338); and yeuree, Y. enterocolitica (88,
L24101).

19

assisting urease activation when tested in vivo, and there was no difference in the
sensitivity of cells containing the truncated UreE versus the wild—type protein to elevated
nickel ion concentrations. These results suggest that that histidine-rich tail is not required
in the urease activation pathway nor does it function to reduce nickel toxicity in the cell.
It is interesting, however, that a nickel transport system has not been detected in any of
the microbes possessing a UreE protein containing a histidine-rich carboxyl terminus
(102). Microbes that contain a nickel permease possess a UreE protein lacking this
histidine-rich motif. Although the role for this histidine-rich tail is unknown, it has been
suggested that it may function in a nickel-storage role (102).

Unlike UreD, the K. aerogenes UreE accessory protein is synthesized at high
levels in recombinant E. coli cells, which has made purification of the protein relatively
facile (72). A single-step purification of the P. mirabilis protein has also been described
(91 ). The cytoplasmic K. aerogenes protein appears to function as a dimer (M; = 35,000)
that binds approximately six nickel ions (Kd ~ 10 uM) as shown by equilibrium dialysis
measurements. The protein is rather specific for binding nickel ions; competition for
approximately one half of the metal-binding sites was observed with 10 uM zinc ions.
Competition with copper and cobalt ions was demonstrated for selected subsites at higher
concentrations. X-ray absorption and variable-temperature magnetic circular dichroism
spectroscopic studies have indicated that UreE contains Ni(II) in pseudooctahedral
geometry liganded with 3-5 histidine imidazole groups and the remainder of the ligands
being nitrogen or oxygen donors (72). Hexagonal bipyramidyl crystals of UreE

apoprotein were obtained; however, these crystals diffract to only 2.9 A and were

terr

“’3:

she

C01

CO

CO

20

observed to fracture upon nickel ion addition, consistent with a conformational change
occurring upon nickel addition.

The truncated K. aerogenes UreE protein lacking the histidine-rich carboxyl
terminus mentioned above has also been purified and partially characterized. The protein
was able to bind to a nickel aflinity resin as observed with wild-type protein and was also
shown to bind two nickel ions per dimer in a cooperative manner (102). Metal
competition experiments using equilibrium dialysis methods indicated that copper
competes for nickel ion binding sites at low concentrations (<50 uM), while cadmium,
copper, and zinc ions compete to a lesser extent.

UreE is likely to function as a nickel donor in metallocenter assembly, possibly
donating nickel to a urease-accessory protein complex (see section ILE); however, direct
proof that nickel binding to UreE is important to metallocenter assembly in urease has not

been obtained.

E. Characterization of UreF

UreF is the least well characterized of the mease accessory proteins. A functional
UreF is absolutely essential for detecting any urease activity in viva, but limited in vitra
activation of urease in extracts of ureF deletion mutants was reported (48). These results
highlight the important difi‘erences that are present between in viva and in vitra Apo
activation. Similar to the findings for UreD and UreE sequences, the UreF sequences are

poorly conserved among the various urease gene clusters (Figure 5), and no significant

 

Fig

an

21

Figure 5. Sequence comparison of selected UreF sequences. The UreF sequences,
analyzed according to (61), are from: kauref, K. aerogenes (19, M36068); pmuref, P.
mirabilis (20, P1709l); ecuref, E.cali (94, L03308); bacuref, Bacillus sp. strain TB-90
(28, D14439); hpuref, H. pylori (55, M84338); and yeuref, Y. enterocolitica (88,

L24101).

 

bacuref
hpuref
yeuref

kauref
pmuref
ecuref
bacuref
hpuref
yeuref

kauref
pmuref
ecuref
bacuref
hpuref
yeuref

kauref
pmuref
ecuref
bacuref
hpuref
yeuref

kauref
pmuref
ecuref
bacuref
hpuref
yeuref

kauref
pmuref
ecuref
bacuref
hpuref
yeuref

51

SQGL..EWAV
SQGL..EWAI
SFGL..ETYI
SFGLLARNLH
SNGV..ESAI

101

GDIAAAQRWT
GDSDTVKYWC
NEPSALVELD
QDLKRILGVE
DDRDGIIRAD

151

PPPWRSLCQQ
DDTLQQRVKQ
NRIKEKKAYG
NAYAQQTEDP
EQIKNGNTAG

201

PFGQQAAQQL
PLGQSAGQKM
PLGQSAGQNI
PIGQTDGQRI
PLSQNDGQKI
RVTHFDTQHI

251

YSRLFRS*
YTRLFRS*
NVRLFMS*
YSRLYMS*
HVRLFSN*

EAGWVLDVAA
EKGWVCSAET
QEKVITDKES
PAKKVTNKES
QTGVVRDVPT

AYLLACRETR
DFMVASRETK
HILEASNVAQ
EIITLSTSPM
WAVNNRKLNE

SQLAGMAWLG
TQLMAFALAA
HSAIVFAIVA
THATSYGVFA
TYPVTQAVVM

ILRLCDHYAA
LEALAEQIPA
LFTLAERIPE
LVEIQPLLEE
LLSLQSPFNQ
LFELNHDIEK

22

PKTDSNAHVD
...... MNAS

FBRWQRRQMT
LSDWLSAQMT
FKNAISVYIR
ALKYLKANLS
LKGFVLTALK

ELREEERNRG
ELRQEERQPG
ETRSGNQRMG
ELRLANQKLG
ESRLMATRMG

VRWRIALPEM
VHWHIDSEKL
YHLKVTKETA
ASLGIELKKA
AAQGIGQREV

EMPRALAAPD
IVELSAHWPQ
VVAQSAIWPL
GVRTISQLPK
LIEKTLELDE
FCDIAEIGDI

QRLRLMQLAS
ADLRLYQLVS
RLLSLFQLCD
NEFLILQVND
DLIRIMQFGD

EGFFTVDLPL
GTLATLELPI
KQLFFTEGLA
SQFLYTEMLS
QA.ASCDGMG

AAFARLLSDW
IAFPRLLPQL
ERMAKLCVDL
NRFIK.TLQA
KKLAEMSIHV

ALSLGYSWIE
CCAYVWGWLE
VGAYLFANVS
LRHYLYAQTS
VVMHQYGVAM

GDIGSATPLA
EDIGSLRQLK
DDIGSFTPAQ
EDLGAVSPGM
SHLCAASVQN
DQMSSYVPIV

50
SNLPVGGYSW
PSLPVGAFTY
SNFPSGSFSH
AVFPIGSYTH
SVLPVGAFTF

100
FARLYRACEQ
LRQLQTSLAK
CILAYEAMEK
LKLTYESALQ
VVAAHRAVVA

150
QPD ...... C
GIE ...... L
YPSPILIEYT
MNELDIGAFF
VEHPLISWWL

200
SAVMAGVKLV
NTVMSGVKLV

250

IIASSRHETQ
EIAQMRHERL
DIKAMQHESL
DVLAAVHVKA

 

 

 

ho

a!

Cl

23

homology was detected between UreF and other proteins found in various data bases. No
significant regions of hydrophobicity are predicted for the protein, consistent with a
probable cytoplasmic location. The UreF protein (Mr = 27,000) is synthesized at low
amounts in the cell (57), making purification and characterization of the protein difficult.
Although the UreF protein has only been partially purified (Chapter 6), a peptide with the
appropriate size and amino-terminal sequence for UreF has been identified as a
component in several protein complexes that are found in cells grown in the absence of
nickel (92, 104). In addition to UreF, one series of complexes (92) includes urease,
UreD, and UreG (termed DFG-Apo, see later). UreF has also been detected in a series of
complexes containing urease and UreD (termed DF-Apo) in ureG deletion mutants (104,
Chapter 4) and in a UreD-UreF-UreG complex in urease deletion mutants (section II.F,
Chapter 5). UreF in the DF-Apo species appears to preclude nickel ions from the urease
active site until Lys-217 is carbamylated, but is unable to preclude other metal ions such
as Co, Cu, and Zn. The precise roles of some of these complexes remains to be
elucidated, but I speculate that UreF functions within several complexes rather than as the

free protein.

F. Characterization of UreG

UreG is the most highly conserved accessory protein in the urease gene cluster
(Figure 6). Four regions of near identity among the reported sequences can be observed:
residues 16-26, 74-82, 112-150, and 157-178 (according to the numbering system shown

in Figure 6). The first of these conserved regions corresponds to a sequence known as a

P-ioot
where
in U
actit:
two i
other
horn-
fmdi
met:
am

the

24

P-loop motif. The P-loop motif is typically found in ATP- and GTP-binding proteins (73)
where it is known to function in nucleotide binding. The presence of this sequence motif
in UreG may be related to the finding of an energy dependence for in viva urease
activation (46). In addition to the extensive regions of similarity within UreG sequences,
two intriguing sequence relationships have been reported between UreG proteins and
other proteins. In one case, the P. mirabilis UreG protein was found to exhibit limited
homology to a 60 kDa chaperonin of the thermophilic bacterium ps-3 (58). Based on this
finding, the authors suggest that UreG may function as a chaperone in the urease
metallocenter assembly pathway. Of greater interest, Wu (74) noted that the K.
aerogenes UreG sequence (19) is approximately 25 percent identical to the sequence of
the E. coli hypB gene product (75). This gene is part of the hydrogenase pleiotropic
operon that is required for activation of the three nickel-containing hydrogenases found in
E. coli. HypB appears to have a role in nickel-ion processing since mutations in the hypB
gene can be complemented by the addition of high levels of nickel ion to the growth
medium (76). Of additional interest, HypB has been purified and found to bind and
hydrolyze GTP (77). The homology between the UreG and HypB sequences is consistent
with the suggestions that UreG may have nucleotide binding capabilities and that urease
and hydrogenase accessory proteins may have descended from a common nickel
metallocenter assembly system. Those hypB sequences that encode proteins containing a
polyhistidine region (see section II. D) may thus represent fusions of UreE-like and UreG-
like proteins.

Protein studies involving UreG have not been reported; however, selected
properties of this protein have been determined. For example, the K aerogenes protein
has been purified and partially characterized by making use of plasmids that express
relatively high levels of ureG in recombinant E. coli cells. The monomeric protein was
found to be located in the cytoplasm by immunogold electron microscopic methods
(Mann Hyung Lee, unpublished), consistent with the lack of significant hydrophobic

(T

'1'

kaureg
pmureg
ecureg
bacureg
hpureg
yeureg

kaureg
pmureg
ecureg
bacureg
hpureg
yeureg

kaureg
pmureg
ecureg
bacureg
hpureg
yeureg

kaureg
pmureg
ecureg
bacureg
hpureg
yeureg

kaureg
pmureg
ecureg
bacureg
hpureg
yeureg

VNSHSTDKRK
51

YTKEDQRILT
YTQEDAKILT
YTQEDAKILT
YTKEDAQFLL

YTKEDAEFMC
VTTEDAKQVK

101

EKFGNLDLIF
MRHKNLDIVF
IRHKNLDIVF
ERHPDLELIF
GRFPNLELLL
ERFPDSNLIM

151

TKSDFLVINK
THPDMMVINK
THSDLLVINK
IKSVLFIINK
TRSDLLVINK
VQADILVINK

201

IIAFLEDKGM
IIDFIIDKGM
IIEFIIDKGM
VLEWIKTNAL
VIAWIKRNAL
LVDMIMRDFL

HPLRVGVGGP
QPLRIGVGGP
QPLRIGVGGP
EPIRIGIGGP
.MVKIGVCGP
KITRIGIGGP

EA..GALAPE
RA..QALDAD
RA..EALDAD
KH..GVLPAD
KN..SVMPRE
RTLKGILDEE

VESGGDNLSA
VESGGDNLSA
VESGGDNLSA
IESGGDNLAA
IESGGSNLSA
IESGGDNLTL

TDLAPYVGAS
IDLAPYVGAS
IDLAPYVGAS
IDLAPYVGAS
IDLAPYVGAD
IDLAPYVGAS

25

VGSGKTALLE
VGSGKTALLE
VGSGKTALLE
VGAGKTMLVE
VGSGKTALIE
VGSGKTAIIE

RIVGVETGGC
RIIGVETGGC
RIIGVETGGC
RVIGVETGGC
RIIGVETGGC
KILGVETGAC

TFSPELADLT
TFSPELADLL
TFSPELADLT
TFSPELVDFS
TFNPBLADFT
TFSPALADFY

LEVMASDTQR
LEVMEADTAK
LEVMEADTAR
LEVMERDTLA
LKVMERDSKK
LDVMESDTKV

222

LGK* ......
LRR*......
LGR* ......
LYGLES*...
LED*....

FTHVQPQGEH A*

ALCKAM.RDT
VLCKAM.RDS
VLCKAM.RDT
KLTRAM.HKE
ALTRHM.SKD
VITPILIKRG

PHTAIREDAS
PHTAIREDAS
PHTAIREDAS
PHTAIREDAS
PHTAIREDAS
PHTAVREDPS

IYVIDVAEGE
FMLIDVAEGE
IYVIDVAEGE
IYIIDVAQGE
IFVIDVAEGD
IYVIDVAEGE

MRGDRPWTFT
MRPVKPYVFT
MRPVKPYVFT
ARGDKPYIFT
IAAKSPLFLP
VRGERPYILT

50
WQLAVVTNDI
YQIAVVTNDI
YQIAVVTNDI
LSIAVVTNDI
YDMAVITNDI
IKPLIITNDI

100
MNLAAVEALS
MNLAAVEBLA
MNLAAVEELA
MNFPAIDELK
MNLEAVEEMH
MNIAAVEEME

150
KIPRKGGPGI
KIPRKGGPGI
KIPRKGGPGI
KIPRKGGQGM
KIPRKGGPGI
KIPRKNGPGL

200
NLKQGDGLST
NLKEKVGLET
NLKKKVGLET
NLKDEIGLAE
NIRAKEGLDD
NCKTGQGIEE

Figure 6. Sequence comparison of selected UreG sequences. The UreG sequences,
compared according to (61), are from the following species: kaureg, K aerogenes (19,
M36068); pmureg, P. mirabilis (58, 221940); ecureg, E.cali (56, 94; L03308); bacureg,
Bacillus sp. strain TB-90 (28, D14439); hpureg, H. pylori (55, M84338); and yeureg, Y.

enterocolitica (88, L24101).

ad:

t‘i:

26

regions in the protein according to sequence analysis. Despite the presence of a P-loop
sequence motif in this protein, purified UreG is unable to bind significant levels of ATP
or GTP as shown by equilibrium dialysis measurements (Chapter 6). Nevertheless, site-
directed mutants that are defective in the P-loop sequence fail to activate urease when
examined in viva. Apo in these site-directed P-loop mutants, similar to the ureG deletion
mutants mentioned earlier, is able to be partially activated when C02 and nickel ions are
added to cell extracts. This result again demonstrates the great distinction between in
viva and in vitra activation of urease (i.e., a functional UreG is crucial for detection of
urease activity in viva whereas partial in vitra activation of Apo can occur without UreG).
Thus, although there is no energy dependence for in vitra activation of the pmified Apo
or D-Apo (38), nucleotide hydrolysis may be required in viva and the nucleotide may bind
to UreG. Rather than binding to the free protein, however, nucleotides may bind to UreG
only when it is present in the DFG-Apo species (see section II.E) or a UreD-UreF-UreG
complex described below. i

In addition to UreG being detected in a series of DFG-Apo complexes, UreG is
also present in an additional complex in urease deletion mutants. A complex containing
UreD, UreF, and UreG (termed DF G) was partially purified and characterized (Chapter
6). This complex binds to an ATP-linked agarose column, while purified UreG does not.
E. coli cells containing a mutant form of UreG, in which the conserved Thr residue in the
P-loop motif was changed to an Ala (T21A), still formed the DFG complex as observed
with wild-type UreG. This indicates that the P-loop is not essential for UreD, UreF, and

UreG complex formation. However, the complex containing T21A did not bind to an

 

 

5X

ior

27
ATP-linked agarose resin which supports the hypothesis that UreG binds nucleotides and

that the Thr in the P-loop motif is essential for nucleotide binding.

G. Current Model for Bacterial Urease Metallocenter Assembly

A working model describing assembly of the urease metallocenter is illustrated in
Figure 7. In nickel-depleted E. coli cells containing the K aerogenes urease gene cluster,
most urease is found as the tree Apo species; however, approximately 10 percent is
present as the D-Apo and a smaller proportion is present as DF-Apo or DFG-Apo (48, 92,
104, Chapter 4). Although the Apo, D-Apo, and DF-Apo can be activated in vitra by
addition of nickel ions and carbon dioxide (38, 48, 104, Chapter 4), these processes do
not appear to take place in viva. Rather, I propose that the DFG-Apo is the species that is
activated in viva. Formation of this complex may occur by a sequential binding of UreD,
UreF, and UreG to the Apo species or by formation of the DFG species with subsequent
binding to the Apo. The lack of in viva activation of Apo, D—Apo, and DF-Apo species
may be related to nickel ion sequestration and limiting levels of carbon dioxide in the cell

[atmospheric C02 concentrations are around 0.03 percent, whereas half-maximal rates of
Apo activation required sevenfold higher C02 levels (38)]. Alternatively, these species
may bind other metal ions in the cell and the inactive metal-substituted proteins must be
processed via DFG-Apo to allow productive binding of nickel ion, as suggested by
experimental results in which Apo, D-Apo and DF-Apo appear to bind a range of metal

ions (101, 104, Chapter 4). Thus, I speculate that the UreD, UreF, and UreG proteins

C0

28

may be involved in delivery of nickel ion and/or carbon dioxide to the Apo species or in
dismantling of non-productive metallocenters in the protein.

As one possibility, the accessory proteins may be required for proper docking or
metal ion transfer between the UreE holoprotein and Apo complex. Although the role of
UreE has not been established, its nickel-binding ability makes it an attractive candidate
as the cellular nickel donor for urease activation (72, 102). As a second possibility, the
UreD, UreF, and UreG proteins may function in generating carbon dioxide within or
delivering C02 to the Apo species. For example, these proteins, as an associated unit,
could have some type of decarboxylase activity, possess carbonic anhydrase activity,
activate C02 possibly via a phosphate intermediate (e. g. H0-C0-0P03), or ferry C02
bound to biotin or other carboxyl group carrier. Whether required for UreE interaction or
"C02" delivery, a role for nucleotide binding and hydrolysis can be postulated, consistent
with the conserved UreG P-loop motif and the in viva activation results. A third
alternative function for the accessory proteins may involve an energy-dependent
expulsion of improperly bound nickel or metal ion to the protein. This role is reminiscent
of that for Rubisco activase (78), and is supported by kinetic and metal quantitation
evidence that nickel can bind to Apo in the absence of C02 to form an inactive species
(3 8, 101). Studies of the DF-Apo complex indicate that the presence of UreF eliminates
the inhibitory binding of nickel ion to non-carbamylated protein but does not, however,
prevent other divalent metal ions from binding and inactivating Apo (104, Chapter 4).
Once activated in the cell, the DFG-Apo, thought to be the physiologically relevant

complex, is thought to dissociate and urease holoprotein is only found as the free enzyme.

29

Figure 7. Model for in viva urease metallocenter assembly. The incorporation of nickel
into urease apoprotein in viva is proposed to occur by a complex process involving four
accessory proteins: UreD, UreE, UreF, and UreG. Nickel enters the cell using a nickel
transport system. UreE (®) functions as the putative nickel donor and delivers nickel
ions to the DFG-Apo complex. The model proposes that Apo either sequentially binds
UreD, UreF, and UreG, or it binds the DF G complex to form the DFG-Apo complex.
Insertion of nickel and C02 into the active site may require nucleotide hydrolysis in
which UreG may play a role. Formation of holourease results in dissociation of the

accessory proteins from the enzyme.

30

Om: gageso

>o=<m Canoe
>UOUBESm

         

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Z.

383%
2_

030233

 

31

H. Accessory Genes Required For Urease Activation In Eukaryotes

Metallocenter accessory genes also appear to be required for urease activation in

eukaryotes. The best-characterized plant urease metallocenter assembly genes identified

to date are in soybean, Glycine max (L.) Merr. Soybean has three urease isozymes (14,
79-82): ubiquitous (leaf), embryo-specific (seed), and a background urease fiom an
associated bacterium Methylabacterium mesaphilicum. Pleiotropic mutations in two
plant loci, Eu2 and Eu3, result in the loss of all urease activity despite normal levels of

urease gene expression (80). The authors suggested that the mutants are defective in a

urease matmation process, such as nickel insertion into the Apo. 63NiCl2 studies

indicate that Eu2 and Eu3 mutants take up normal amounts of NiCl2, and grafting

experiments indicate that transport of nickel through mutant roots and stems is
unafl'ected. These experiments suggest that mutations in Eu2 and Eu3 have no effect on
nickel ion uptake or transport, but somehow affect nickel incorporation into the
metallocenter (80). It is surprising that mutations in soybeans at these two loci eliminate
urease activity in the phylloplane-associated bacterium (79). Bacteria isolated from
soybeans having these mutations lack both urease and hydrogenase activity, but these
enzymatic activities can be restored by nickel addition to the isolated cultures.

An obvious interpretation of these results is that the plant mutants cause the
bacteria to be deficient in nickel and unable to produce active urease and hydrogenase. It
is possible that the soybean mutants somehow sequester nickel and prevent activation of
all three urease isozymes. The two soybean loci have not been sequenced and their
relationships to the bacterial accessory genes have not been established at this time.
Partial sequences of unidentified genes from Arabidapsis thaliana (GenBank accession
number 218230) and barley, Hordeum vulgare L. (Brian Forde, personal

32

commrmication), which could be related to urease activation in plants, have sequence
similarities to the UreG protein of K aerogenes. Fungal species also have accessory
genes that are required for functional urease activity. Four loci have been identified as
being necessary for functional urease in Aspergillus nidulans (83), Neuraspara crassa
(84), and Schizasaccharomyces pambe (85). In the case of A. nidulans, one of the four
loci, ureA, encodes a urea-specific transport protein. The structural subunit of urease is
encoded by the ureB gene. Another locus, ureC, is required, but its ftmction is unknown.
Finally, the ureD gene is thought to be required for the synthesis or incorporation of the
nickel cofactor based on the ability to correct a mutation in ureD by growing the fungus
in the presence of 0.1 mM nickel sulfate (86). The fungal ureD gene has also been found
to have sequence similarity to the UreG protein of K. aerogenes (Gareth Wyn Grifl'rth,
personal communication). Further research will most likely reveal other relationships
between prokaryotic and eukaryotic accessory proteins as is seen with the urease enzyme

itself.

III. SUMMARY

Metallocenter assembly in urease is clearly a complex and highly specific process.
More extensive research has been done in prokaryotes than in eukaryotes since it is easier
to grow and manipulate bacteria than most eukaryotes. In addition, the fact that urease is
a virulence factor in some bacteria makes understanding bacterial urease activation an
important facet of medical research. Although the players in urease metallocenter
assembly have been identified, their precise roles and order of appearance in this drama
have not yet been clearly identified. UreD may function as a chaperone or molecular pr0p
that maintains a state of urease that has enhanced ability to be activated, but how the D-

Apo and related complexes are formed, how their structures compare to the native urease

33
structure, how UreD plays this role, and how UreD is recycled are not understood. UreE

is a nickel-binding protein, at least in K aerogenes, but it remains unclear whether its role
in metallocenter assembly is that of a nickel donor. The roles of UreF and UreG in urease
activation are very poorly understood, although the presence of UreF in the DF-Apo
complex prevents non-productive nickel ion-binding by the noncarbamylated urease
species. We can only speculate as to the possible fimction for DFG-Apo species although
it is thought to be the key complex in vivo. The putative P-loop motif in UreG may be
consistent with a role for nucleotides during in viva activation which is supported by the
binding capabilities of the DFG complex to a nucleotide-linked resin. Hopefully some of
what we learn about metallocenter assembly in urease may be applicable to activation

processes in other metalloproteins.

IV. THESIS OUTLINE

The following chapters describe my studies on Klebsiella aerogenes urease
apoprotein (Apo) and h010protein, accessory proteins, and several Apo-accessory protein
complexes. In Chapter 2, I describe crystalline urease activity and related salt inhibition
studies. These studies showing that urease crystals are essentially inactive were
combined with X-ray crystallographic data analysis by Evelyn Jabri and preliminary
crystal activity studies by Louis G. Horn and published in Protein Science 4, 2234-2235
(1995) (103). In Chapter 3, I describe the first demonstration of in vitro activation of

urease apoprotein. The in vitra activation process in cell extracts is slow and the highest

34
specific activity studies (30-40 U/mg protein) only account for activation of

approximately 10% of the Apo that is present. This work was combined with UreD-
urease (D-Apo) studies by Il-Seon Park and published in Proceedings of the National
Academy of Sciences USA 91, 3233-3237 (1994) (48). Chapter 4 describes the
identification and characterization of a series of complexes containing UreD, UreF, and
Apo (DP-Apo) formed in cells that overexpress ureD and ureF. These complexes exhibit
activation properties that are distinct fiom Apo and D-Apo complexes. Unlike Apo and
D-Apo, the DF-Apo complexes are resistant to inactivation by NiC12, exhibit a decreased
bicarbonate concentration dependence, and UreD is masked, presumably by UreF, in
these complexes. I propose that binding of UreF modulates the D-Apo activation
properties by excluding nickel ions from the binding site rmtil after formation of the
carbamylated lysine metallocenter ligand. These studies were published in the Journal of
Bacteriology 178, 5417-5421 (1996) (104). In Chapter 5, I describe the purification of
UreG and the identification of a UreD-UreF-UreG (DFG) complex. Purified UreG does
not bind or hydrolyze nucleotides, contain biotin, or exhibit carbonic anhydrase activity.
Site-directed mutagenesis of two residues in the UreG P-loop motif, known to be
essential for nucleotide binding in several proteins (73 ), are identified as essential for in
viva activation of Apo. Unlike purified UreG, the DFG complex binds to an ATP-linked
agarose resin. In cells containing the UreG P-loop variants, the DFG complex is still
formed but doesn’t bind to an ATP-linked resin. This suggests that the P-loop motif is
not essential for DF G complex formation, but is essential for nucleotide binding. In

Chapter 6, I describe the overexpression of ureF and the high insolubility of UreF in the

35
absence of the other urease gene products unless fused to thioredoxin. In the presence of

the other urease genes, a DFG-Apo complex (termed DFG-Apo*) distinct fi'om those
described earlier (92) is produced and exhibits properties similar to Apo and D-Apo.
Chapter 7 describes the identification of a series of Apo forms purified from a ureD
deletion mutant that collapse upon incubation with 10 mM dithiothreitol. These species
are not formed in a urease C319A ureD deletion mutant but instead form species that
migrate in a manner that is coincident to forms identified when DF-Apo complexes are
activated in vitro. This suggests that C319, the only Cys located in the active site, may
play a role in formation of these species. I also include studies where Apo was incubated
with DFG complex to test the urease metallocenter model proposal that DF G can bind to
Apo to flour DFG-Apo. A band similar in size to DFG-Apo was identified by
irnmunoblot analysis supporting the hypothesis that the DFG complex might form first
with subsequent addition of Apo to form DFG-Apo. In Chapter 8, I summarize the
current model for urease metallocenter assembly and discuss future prospects. As an
appendix to the thesis, I include a manuscript that I co-authored for which I supplied
urease and seleno-methionine-containing urease to Evelyn Jabri and Dr. Andy Karplus at
Cornell University in Ithaca, New York. This work describes the crystal structure of

urease from Klebsiella aerogenes and was published in Science 268, 998-1004 (1995).

10.

ll.

12.

13.

36
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CHAPTER 2

UREASE ACTIVITY IN THE CRYSTALLINE STATE

The studies reported here were combined with X-ray crystallographic data analysis by
Evelyn Jabri and preliminary crystal activity studies by Louis G. Horn and published as
Moncrief, M. B. C., L. G. Horn, E. Jabri, P. A. Karplus, and R. P. Hausinger. (1995).
Urease activity in the crystalline state. Protein Science, 4:2234-2236. Information that
was available as an electronic appendix to the Protein Science paper is integrated into this

text.

46

47
ABSTRACT

Crystalline urease from Klebsiella aerogenes was found to have less than 0.05%
of the activity observed for the soluble enzyme under standard assay conditions. Li2$04,
present in the crystal storage buffer at 2 M concentration, was shown to inhibit soluble
urease by a mixed inhibition mechanism (Ki’s of 0.38 i 0.05 M for the free enzyme and
0.13 :1: 0.02 M for the enzyme-urea complex). However, the activity of crystals was less
than 0.5% of the expected value for the salt content in the crystal storage buffer,
suggesting that salt inhibition does not account for the near absence of crystalline activity.
Dissolution of crystals resulted in approximately 43% recovery of the soluble enzyme
activity, demonstrating that protein denaturation during crystal growth does not cause the
dramatic diminishment in the catalytic rate. Finally, crushed crystals exhibited only a
three-fold increase in activity over that of intact crystals, indicating that the rate of
substrate difi‘usion into the crystals does not significantly limit the enzyme activity.
Urease is efl‘ectively inactive in this crystal form and the small amounts of activity
observed likely arise from limited enzyme activity at the crystal surfaces or trace levels of

enzyme dissolution into the crystal storage buffer.

48
INTRODUCTION

Urease (EC 3.5.1.5) is a nickel-containing enzyme (1) that catalyzes the hydrolysis
of mm to yield ammonia and carbamate. Carbamate is unstable and spontaneously
decomposes to yield a second molecule of ammonia and carbonic acid. The high levels
of ammonia generated by these reactions and the resulting elevation of pH have important
ramifications for medicine and agriculture (reviewed in 2). For example, urease is a
virulence factor in pathogenic bacteria that cause gastric ulceration, urinary stone
formation, pyelonephritis, and other dysfunctions. Moreover, urea-based fertilizers are
rapidly decomposed by soil urease activity with consequences that include phytopathicity
and loss of volatilized nitrogen.

Urease from the soil bacterium Klebsiella aerogenes has been crystallized (3) and
the three dimensional structure has been determined (4). The structure shows that urease
is a tightly associated ((113103 trimer (or-60.3 kDa, B-11.7 kDa, y-ll.1 kDa) containing
three active sites per holoenzyme and two nickel ions per active site. Thus far no binding
of substrate or inhibitors soaked into the crystals has been observed. These failures raise
the question of whether urease has activity in the crystalline state. This study
demonstrates that crystalline urease is essentially inactive and examines the possible

factors to account for the diminishment in activity.

49
MATERIALS AND METHODS

Enzyme purification and crystallization. Urease was purified fi'om K.
aerogenes CG 253 cells carrying pKAU19, a plasmid containing the K aerogenes urease
gene cluster, that were grown at 37 °C in MOPS-glutamine media as described previously
(5). Urease crystals were grown by published procedures using the hanging drop method
(3). Crystals were stored in crystal storage buffer containing 2M Li2SO4 and 100 mM
HEPES (pH 7.75) and were used within 3.5 weeks after they were set up for growth. All
experiments reported in this paper were carried out with protein fi'om a single

preparation.

Assays. Urease activity was measured by quantitating the rate of ammonia release
from urea by formation of indophenol, which was monitored at 625 nm (6). One unit of
urease activity was defined as the amount of enzyme required to hydrolyze 1 mole of
urea per minute at 37 °C. The standard assay buffer consisted of 25 mM HEPES (pH
7.75), 0.5 mM EDTA, and 50 mM urea. When testing whole and crushed crystal activity
the assay buffer additionally included 2 M Li2SO4 and the solution was gently agitated on
a platform shaker. All assays with crystals were carried out at 30 °C and the
experimentally determined values were adjusted by a factor of 1.4 to provide 37 °C
values. Whole crystals were cubes of 230-250 pm on each edge and crushed crystals had
fi'agments of < 30 pm in the greatest dimension. For crystal dissolution, crystals were

washed with crystal storage buffer, dissolved in 500 pl water, and immediately assayed.

50

All assays were linear over the times examined. The protein content of soluble urease
was determined by the method of Lowry et al. (7) using bovine serum albumin as the
standard. The protein content of urease crystals was determined by using the following

equation:

size (um)3 x unit cell x 24 0437 units x 1 mole 0437 x 83,202 g
crystal (1.71 x 10-2 “my unit cell 6.023 x 1023 (107 units 1 mole (107

 

 

Using this equation a typical (230-250 um)3 crystal contains 8.09-10.4 pg of urease.

Salt inhibition of urease activity. Since the crystal storage buffer contained 2 M
Li2SO4, we tested the activity of urease in the presence of varied concentrations of
Li2SO4, LiCl, or Na2SO4, and various concentrations of urea. Kinetic data for each salt
concentration were fit by using the method of Wilkinson (8) and subsequent replots of
slopes and intercepts for analysis of the inhibition constants used well established

methods (9).

RESULTS AND DISCUSSION

Activity of crystalline urease. Whereas fieshly prepared K. aerogenes urease
possessed a specific activity of 3,500 units/mg, the activity observed for crystalline
enzyme in crystal storage buffer was only 1.54 units/mg (Table 1). Four possible reasons
to account for the low activity in the urease crystals were considered: (i) a component in

the crystal storage buffer may inhibit the enzyme, (ii) the crystals may represent an

51

Table 1. Specific activities of soluble and crystalline K aerogenes urease

 

 

 

 

Sample Specific Activity (units/mg)__
2 M Li2SO4 No salt
Soluble enzyme 321 i 10 3,500 :l: 100
Crystalline enzyme 1.54 :1: 0.25 -
Crushed crystalline enzyme 4.3 i 0.7 -
Dissolved crystalline enzyme - 1,500 i 140

 

52
irreversibly denatured and inactive state of the enzyme, (iii) the rate of substrate diffusion

into the crystals may be limiting such that only the molecules exposed to the surface are
active, and (iv) the crystalline enzyme is restricted from undergoing conformational
changes required for catalysis. In the latter case, the observed levels of activity in crystals
may correspond to enzyme molecules at the crystal surface and/or soluble enzyme arising
fi'om slight dissolution of the crystal in fresh crystal storage buffer. Experimental
evidence related to these considerations are described below.

To examine the first possibility, soluble urease was added to buffer containing 2
M Li2SO4 to simulate crystal storage conditions. Under these conditions the enzyme
possessed roughly 320 units/mg. Although Li2SO4 does inhibit urease (see detailed
kinetics below), the approximately ll-fold decrease in activity falls far short of the 2,300-
fold decrease seen in the crystals. The only other component in the crystal storage bufi‘er
is 100 mM HEPES which is known to not interfere with the urease assay. Although it is
possible that atmospheric components (e. g., COz/HCO3' ) or other unidentified factors
may bind to the crystalline enzyme and reduce its activity, such factors would have to
dissociate upon crystal dissolution. The active site in fresh crystals does not appear to
have any tmexpected ligands bound.

With regard to the second possibility, growth of urease crystals of suitable size for
these studies required approximately three weeks at room temperature, thus it is
conceivable that the enzyme denatured during this time and the crystals represented
inactive protein. To address this concern, the urease activity of dissolved crystals was

assessed. As shown in Table I, the specific activity of dissolved crystals was 1,500

53
units/mg, representing a recovery of 43% of the activity of flesh soluble urease. Thus,

protein denaturation during crystal grth does not account for the near absence of urease
activity in the crystalline state.

Although protein crystals are highly hydrated, limited rates of substrate difl‘usion
into crystals have been shown to account for the apparent low specific activities of
selected crystalline enzymes (10). Given the small size of the urea molecule, this process
is not likely to be significant in the case of urease. Nevertheless, I attempted to test this
possibility by observing the effect on activity of crushing individual crystals in their
crystal storage bufier to form fragments where the longest dimension was less than 12.5%
the size of the intact crystal. Only a slight (3-fold) increase in activity was noted (Table
I), possibly arising from enhanced levels of dissolution of enzyme from the increased
surface area of the crystal matrix into fresh crystal storage buffer. Thus, substrate
diffusion into the crystals is not the limiting process to explain the low tu'ease activities in
the crystalline state.

Based solely on ruling out other possible explanations, the low level of urease
activity in the crystalline state probably arises primarily fi'om conformational restrictions
in the crystal lattice. Indeed, I conclude that urease in this crystal form is essentially
inactive. The low levels of activity observed in assays of the crystals are likely derived
from trace amounts of enzyme equilibrating with the solution. Consistent with this
proposal, crystal storage bufler from which a urease crystal had been removed was shown

to possess significant levels of urease activity.

54

Kinetic analysis of salt inhibition. Because urease inhibition by Li2SO4 was
fairly extensive, we examined it in more detail. Inhibition was not restricted to the
lithium cation or the sulfate anion as similar inhibition was observed with Li2SO4, LiCl,
and Na2SO4 (Fig. l). The inhibition is not simply due to ionic strength effects; e.g., the
various concentrations of LiCl are more effective than equivalent concentrations of
Na2S04. In order to characterize the mechanism of inhibition, urease activity was
measured over a range of substrate and Li2SO4 concentrations. A subset of the data is
depicted in a double-reciprocal plot in Fig. 2A. Intersection of the lines below the x-axis
is consistent with a mixed type inhibition where the inhibitor binds to both free enzyme
and the enzyme-substrate complex with dissociation constants of K,- and K,’, respectively
(9). This type of inhibition mechanism can be represented by the following equation:

V

max

state]

 

v:

The values of K, and K,’ were obtained by replots of the slopes and the vertical axis
intercepts, as shown in Fig. 2B and 2C. The K, value of 0.38 d: .05 M and the K; value of
0.13 :l: .02 M indicate that Li2SO4 binds more tightly to the enzyme-substrate complex
than to the free enzyme. The crystal structure gives no evidence for sulfate binding sites
near the catalytic center. Since lithium ions only have 2 electrons, they would not be

easily visible in the crystal structure.

55

100

 

 

 

 

 

80 -
8
5. 60 - 0
'5
'5
3
9
2g 40 j .
0
a.
in

O
20 - .
0 I T I
0.0 0.5 1.0 1.5 2.0
[88"] (M)

F igtu'e l. [Urease activity as a function of salt concentration. Ptuified urease was assayed
at 37 °C in the presence of 25 mM HEPES (pH 7.75), 0.5 mM EDTA, 50 mM urea, and

the indicated concentrations of Li2SO4 (O), Na2SO4 (O), and LiCl (I).

A 204

1.5 -‘

llv (min*mglumole N113)

 

 

 

 

O

 

Vertical axla Intercept

 

 

I
-0.2 «0.1 0.1 0.2 0.3 0.4 0.5

[142804]

Figure 2. Kinetic analysis of Li2804 inhibition of urease. (A) An inverse plot of urease
activity in the presence of Li2SO4 at 0 (O), 0.01 M (O), 0.05 M (I), 0.25 M (A), 0.3 M
([1), and 0.5 M (A) concentrations. (B) Replot of slopes versus [Li2804]. (C) Replot of

vertical axis intercepts versus [Li2SO4].

10.

57
REFERENCES

Hausinger, R. P. (1993). Urease. In Biochemistry af Nickel, pp. 23-57. New
York: Plenum Publishing Corp.

Mobley, H. L. T. & Hausinger, R. P. (1989). Microbial ureases: significance,
regulation, and molecular characterization. Microbiological Reviews 53, 85-108.

Jabri, E., Lee, M. H., Hausinger, R. P. & Karplus, P. A. (1992). Preliminary
crystallographic studies of urease from jack bean and from Klebsiella aerogenes.
Journal of Molecular Biology 227, 934-937.

Jabri, E., Carr, M. B., Hausinger, R. P. & Karplus, P. A. (1995). The crystal
structure of urease from Klebsiella aerogenes. Science 268, 998-1004.

Mulrooney, S. B., Pankratz, H. S. & Hausinger, R. P. (1989). Regulation of
gene expression and cellular localization of cloned Klebsiella aerogenes (K.
pneumaniae) urease. Journal of General Microbiology 135, 1769-1776.

Weatherburn, M. W. (1967). Phenol-hypochlorite reaction for determination of
ammonia. Analytical Chemistry 39, 971-974.

Lowry, O. H., Rosebrough, N. J., Farr, A. J. & Randall, R. J. (1951). Protein
measurement with the F olin phenol reagent. Journal of Biological Chemistry 193,
265-275.

Wilkinson, G. N. (1961). Statistical estimations in enzyme kinetics. Biochemical
Journal 80, 324-332.

Dixon, M. & Webb, E. C. (1961). Enzymes, 3rd Ed. New York: Academic Press,
Inc.

Makinen, M. W. & Fink, A. (1977). Reactivity and cryoenzymology of enzymes
in the crystalline state. Annual Review of Biophysics and Bioengineering 6, 301-
343.

CHAPTER 3

FIRST DEMONSTRATION OF IN VITRO ACTIVATION OF UREASE

APOPROTEIN

The studies described here were combined with D-Apo studies by Il-Seon Park and
published as Park, I.-S., M. B. Carr, and R. P. Hausinger (1994). In vitro activation of
urease apoprotein and role of UreD as a chaperone required for nickel metallocenter

assembly. Proceedings of the National Academy of Sciences USA, 91: 3233-3237.

58

59
ABSTRACT

The formation of active urease in Klebsiella aerogenes requires the presence of

three structural genes for the apoprotein (ureA, ureB, and ureC), as well as four accessory
genes (ureD, ureE, ureF, and ureG) that are involved in functional assembly of the
metallocenter in this nickel-containing enzyme. Here I report the first evidence that
urease apoprotein can be activated in vitro in highly concentrated cell extracts. Slow and
partial activation of urease apoprotein was observed after addition of nickel ions to
extracts of Escherichia coli cells bearing a plasmid containing the K aerogenes urease
gene cluster or derivatives of this plasmid with deletions in ureE, ureF, or ureG. In
contrast, extracts of cells containing a ureD deletion derivative failed to generate active

urease, thus highlighting a key role for UreD in the metallocenter assembly process.

60
INTRODUCTION

Urease (EC 3.5.1.5) is a nickel-containing enzyme that catalyzes the hydrolysis of
mea to form carbonic acid and two molecules of ammonia. Until recently, the best
characterized urease was that from jack bean (1). This enzyme was the first ever
crystallized (2) and the first shown to possess nickel (3). However, because of the ease of
cell growth and genetic manipulation in bacteria compared to plants and the medical
importance of bacterial urease, our understanding of the prokaryotic enzyme has outpaced
knowledge of the plant enzyme (4). Irnportantly, the active site residues of plant and
bacterial ureases are highly conserved and both enzymes possess a novel bi-nickel
metallocenter.

Assembly of the urease bi-nickel metallocenter appears to be a complex process
requiring the action of several accessory gene products. For example, deletion analysis of
the IGebsiella aerogenes urease gene cluster revealed that one gene (ureD) located directly
upstream and three genes (ureE, ureF, and ureG) found directly downstream of the three
genes (ureA, ureB, and ureC) encoding the subunits of the bacterial urease are involved in
the functional assembly of the urease metallocenter (5, 6). Homologs of the accessory
genes have been found in several bacterial species (e.g., 7-9) and an essential role for
auxiliary genes in forming active urease has been confirmed in these and many other
bacteria. Genetic studies are consistent with a similar requirement for mease accessory
genes in the fungi Neurospora crassa (10), Schizasaccharamyces pambe (11), and

Aspergillus nidulans (12, 13). Finally, two loci (Eu2 and Eu3) that appear to encode genes

61
associated with urease maturation factors such as nickel emplacement have been identified

in soybean plants (14). Thus, accessory genes involved in metallocenter assembly may be a
universal component of urease activation.

The detailed roles for the urease accessory genes are unknown. The histidine-rich
K aerogenes UreE protein has been purified and shown to reversibly bind six nickel ions
per dimer (15); hence, it is reasonable to suggest that UreE serves as the nickel donor to
urease apoprotein. UreG contains a P-loop motif that is formd in many nucleotide-binding
proteins (16); thus, this protein may be involved in coupling nucleotide hydrolysis to the
assembly process. In this regard, in viva evidence is consistent with an energy dependence
for urease activation (17). Furthermore, the sequence of K aerogenes UreG was shown
(18) to be approximately 25% identical to that of the Escherichia coli hypB gene product
(19). The hyp operon (hydrogenase pleiotropic operon) is required for ftmctional activation
of hydrogenase. A mutation in hypB was shown to be complemented, in part, by the
addition of high levels of nickel (20), consistent with a role for this protein in nickel
processing. 0f potential significance to the ftmction of UreG, purified HypB protein has
been shown to bind guanine nucleotides and to hydrolyze GTP (21). In contrast to UreE
and UreG, the sequences for UreD or UreF offer no insight into their function and these
proteins have not been purified or characterized. Here 1 demonstrate the ability to partially

activate urease apoprotein in vitro.

MATERIALS AND METHODS

62
Cell Growth and Disruption. E. coli DH5 or DHl carrying pKAU17 (22) or

AureD-Z, AureE-I, AureF, or AureG—I deletion derivatives of pKAUl7 (6) were gown in
Luria-Bertani (LB) medium to late stationary phase. The harvested cultures were
resuspended in 20 mM phosphate (pH 7.0), 1 mM EDTA, and 1 mM 2-mercaptoethanol
bufi‘er and disrupted by three passages through a French pressure cell (American Instrument
Co., Silver Spring MD) at 18,000 lb/inz. Cell extracts were obtained after centrifugation
(100,000 x g for 60 min) at 4°C. The samples were further sterilized by passage through a

0.2 pm filter into sterile tubes.

Urease Activity Assays. Urease activities were assayed in 25 mM HEPES (pH
7.75) and 0.5 mM EDTA bufi'er containing 50 mM urea. Linear regession analysis of the
released ammonia, determined by conversion to indophenol (23), versus time yielded initial
rates. One unit of activity is defined as the amount of enzyme required to degrade 1 pmol
urea per min at 37°C. Protein concentrations were determined by a commercial assay (Bio-

Rad, Hercules, CA) with a bovine serum albumin standard.

RESULTS AND DISCUSSION

In Vitra Activation of Urease Apoprotein. E. coli (pKAU17) carries the wild-type
K aerogenes urease gene cluster. The urease apoprotein (Apo) in extracts fiom these cells
(gown in the presence of nickel-free medium) is partially activated by incubation with 1

mM NiCl2 (Fig. 1, 0). Although in viva activation of Apo has been described [e.g., (17)],

63
the data shown in Fig. 1 represent the first evidence for in vitro activation of this enzyme.

Similar to the in viva situation, the in vitro activation process is very slow, in this case
requiring over a day to reach its limiting activity. The highest specific activity values
observed in these studies (30—40 U/mg protein) account for activation of only about 10% of
the urease that is present [i.e., when gown in the presence of 1 mM NiCl2 E. coli(pKAU17)
cell extracts typically possess specific activity values of 300-400 U/mg (17)]. The rate and
extent of activation is not affected by Apo stability as demonstrated by studies in which
extracts were preincubated at 37 °C for 23 h prior to nickel addition with only partial loss of
competence for activation. Similarly, the leveling off of activity is not due to simultaneous -
formation and inactivation of active enzyme because purified urease holoprotein is
completely stable under the incubation conditions used for activation of the cell extracts
(Fig. 2). Alteration of the activation conditions (varying the pH, bufler composition, ionic
strength, and NiCl2 concentration) failed to improve the extent of activation and the process
was hindered by elevated nickel concentrations (4 mM) and high (> 8.0) or low (< 7.0) pH.
In contrast to the in viva urease activation results reported earlier (17), the in vitro activation
process described above does not appear to require energy. Dialysis of cell extracts prior to
nickel addition or inclusion of apyrase 15 min prior to the activation assay has no efi‘ect on
the process. Further, inclusion of up to 1 mM magresium and 1 mM ATP or GTP does not
significantly alter the activation behavior. Higher concentrations of these nucleotides
appear to inhibit activation, perhaps by sequestering nickel ion.

Analogous Apo activation experiments were carried out using extracts fi'om cells

that were defective in three of the urease accessory genes, ureD, ureF, and ureG (Fig. 1).

40-
35-1
30-
25-
20-

15-

1

0 10 20 30 4O 50 60 70

Specific Activity (Ulmg)

 

Time (hours)

Figure 1. In vitro activation kinetics of urease apoprotein. Cell extracts (2 mg/ml) from
E. coli cells carrying pKAU17 (O), or AureD (I), AureF (V), or AureG (A) deletion
derivatives of this plasmid were incubated in 20 mM phosphate buffer (pH 7.8) containing
1 mM EDTA, 1 mM 2-mercaptoethanol, and 1 mM NiC12 at 37 °C. Aliquots were

withdrawn and assayed for urease activity and protein at the time points shown.

65

[UreE is known not to be essential for urease activation in viva (6), hence, detailed in vitro
activation studies were not carried out with the AureE mutant. Preliminary experiments did
verify the absence of a requirement for UreE in vitro (data not shown)]. Surprisingly,
deletion of ureG (Fig. 1, A) has no inhibitory efiect on the in vitro activation process and
may lead to an increase in the rate and extent of functional metallocenter assembly. This
finding is intriguing because AureG cultures possess no detectable urease activity even
when provided with 1 mM nickel ion in the gowth medium (6). Indeed, the in vitro
activation behavior (starting from no detectable activity and increasing at a rate and to a
level similar to the control extracts) is identical for AureG extracts when obtained from cells
gown with or without nickel ion. The cell extracts fiom a ureF deletion mutant also
exhibit activation competence (Fig. l, Y), but the final specific activity value reached is
significantly depressed compared to the control. Again, the observation of any urease
activity is surprising because AureF cultures have no detectable activity even when
provided with high concentrations of nickel in the gowth medium. Of greatest
significance, Apo in cell extracts from a AureD mutant (gown in the presence or absence of
nickel ion) was not competent for activation under these activation conditions.

In summary, these results provide the first evidence for in vitro activation of Apo
and highlight a key role for UreD in the urease activation process. [Note: These
experiments were carried out prior to the demonstration that CO2 is required for in vitro
activation (24). No bicarbonate was provided in the activation bufl'er, so the only CO2
available was that which diflhsed into the buffer from the atmosphere. This explains, in

part, the very slow kinetics of activation observed in these studies. The UreD-Apo and

66
UreD-UreF-Apo complexes can better incorporate trace levels of CO2 than Apo, so the

activation reported here is likely to arise predominantly from Apo that is present as

complexes with accessory proteins]

 

 

 

1400
E
a
E !---O
< 1200 -+
i»
1000 u r T I I l
0 20 40 60 80 100 120

Time (hours)

Figure 2. Urease stability during incubation at 37 °C. Purified holourease was incubated
in 20 mM phosphate (pH 7.3), 1 mM EDTA, and 1 mM 2-mercaptoethanol at 37 °C and

assayed for activity at the indicated timepoints.

10.

11.

67

REFERENCES

Zemer, B. (1991). Recent advances in the chemistry of an old enzyme, urease.
Bioarganic Chemistry 19, 116-131.

Sumner, J. B. (1926). The isolation and crystallization of the enzyme urease.
Journal of Biological Chemistry 69, 43 5-441.

Dixon, N. E., Canola, C., Blakeley, R. L. & Zemer, B. (1975). Jack bean
urease (EC 3.5.1.5). A metalloenzyme. A simple biological role for nickel?
Journal of the American Chemical Society 97 , 4131-4133.

Mobley, H. L. T. & Hausinger, R. P. (1989). Microbial ureases: sigrificance,
regulation, and molecular characterization. Microbiological Reviews 53, 85-108.

Mulrooney, S. B. & Hausinger, R. P. (1990). Sequence of the Klebsiella
aerogenes urease genes and evidence for accessory proteins facilitating nickel
incorporation. Journal of Bacteriology 172, 5837-5843.

Lee, M. H., Mulrooney, S. B., Renner, M. J., Markowicz, Y. & Hausinger, R.
P. (1992). Sequence of ureD and demonstration that four accessory genes (ureD,

ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis. Journal
of Bacteriology 174, 4324-43 30.

Cussac, V., Ferrero, R. L. & Labigne, A. (1992). Expression of Helicabacter
pylori urease genes in Escherichia coli gown under nitrogen-limiting conditions.
Journal of Bacteriology 174, 2466-2473.

Jones, B. D. & Mobley, H. L. T. (1989). Proteus mirabilis urease: nucleotide
sequence determination and comparison with jack bean urease. Journal of
Bacteriology 171, 6414-6422.

Collins, C. M. & Gutman, D. M. (1992). Insertional inactivation of an
Escherichia coli urease gene by IS341]. Journal of Bacteriology 174, 883-888.

Benson, E. W. & Howe, H. B., Jr. (1978). Reversion and interallelic
complementation at four urease loci in Neurospora crassa. Molecular and
General Genetics 165, 277-282.

Kinghorn, J. R. & Fluri, R. (1984). Genetic studies of purine breakdown in the
fission yeast Schizasaccharomyces pambe. Current Genetics 8, 99-105.

12.

l3.

14.

15.

16.

17.

18.

19.

20.

21.

22.

68

Mackay, E. M. & Pateman, J. A. (1982). The regulation of urease activity in
Aspergillus nidulans. Biochemical Genetics 20, 763-776.

Mackay, E. M. & Pateman, J. A. (1980). Nickel requirement of a urease-
deficient mutant in Aspergillus nidulans. Journal of General Microbiology 116,
249-25 1.

Meyer-Bothling, L. E., Polacco, J. C. & Cianzio, S. R. (1987). Pleiotropic
soybean mutants defective in both urease isozymes. Molecular and General
Genetics 209, 432-438.

Lee, M. H., Pankratz, H. S., Wang, 8., Scott, R. A., Finnegan, M. G.,
Johnson, M. K., Ippolito, J. A., Christianson, D. W. & Hausinger, R. P.
(1993). Pruification and characterization of Klebsiella aerogenes UreE protein: a
nickel-binding protein that functions in urease metallocenter assembly. Protein
Science 2, 1042-1052.

Saraste, M., Sibbald, P. R. & Wittinghofer, A. (1990). The P-loop-a common
motif in ATP- and GTP- binding proteins. Trends in Biochemical Sciences 15,
430-434.

Lee, M. H., Mulrooney, S. B. & Hausinger, R. P. (1990). Purification,
characterization, and in vivo reconstitution of Klebsiella aerogenes urease
apoenzyme. Journal of Bacteriology 172, 4427-4431.

Wu, L.-F. (1992). Putative nickel binding sites of microbial proteins. Research in
Microbiology 143, 347-351.

Lutz, A., Jacobi, A., Schlensog, V., Bfihm, R., Sawers, G. & Bock, A. (1991).
Molecular characterization of an operon (hyp) necessary for the activity of the
three hydrogenase isoenzymes in Escherichia coli. Molecular Microbiology 5,
123-135.

Waugh, R. & Boxer, D. H. (1986). Pleiotropic hydrogenase mutants of
Escherichia coli K-12: gowth in the presence of nickel can restore hydrogenase
activity. Biochimie 68, 157-166.

Maier, T., Jacobi, A., Sauter, M. & Bock, A. (1993). The product of the hypB
gene, which is required for nickel incorporation into hydrogenases, is a novel
guanine nucleotide-binding protein. Journal of Bacteriology 175, 630-635.

Mulrooney, S. B., Pankratz, H. S. & Hausinger, R. P. (1989). Regulation of
gene expression and cellular localization of cloned Klebsiella aerogenes (K
pneumaniae) urease. Journal of General Microbiology 135, 1769-1776.

23.

24.

69

Weatherburn, M. W. (1967). Phenol-hypochlorite reaction for determination of
ammonia. Analytical Chemistry 39, 971-974.

Park, I.-S. & Hausinger, R. P. (1995). Requirement of carbon dioxide for in
vitro assembly of the urease nickel metallocenter. Science 267 , 1156-1158.

CHAPTER4

PURIFICATION AND ACTIVATION PROPERTIES OF URED-UREF-UREASE

APOPROTEIN COMPLEXES

These studies were published in the Journal of Bacteriology, 178: 5417-5421, 1996.

70

71

ABSTRACT

In viva assembly of the Klebsiella aerogenes urease nickel metallocenter requires
the presence of UreD, UreF, and UreG accessory proteins and is further facilitated by UreE.
Prior studies had shown that urease apoprotein exists in an tmcomplexed form as well as in
a series of UreD-urease (I.-S. Park, M. B. Carr, and R. P. Hausinger, Proc. Natl. Acad. Sci.
USA 91:3233-3237, 1994) and UreD-UreF-UreG-urease (I.-S Park, and R P. Hausinger, J.
Bacteriol. 177:1947-1951, 1995) apoprotein complexes. This study demonstrates the
existence of a distinct series of complexes comprised of UreD, UreF, and urease apoprotein.
These novel complexes exhibited activation properties that were distinct from urease and
UreD-urease apoprotein complexes. Unlike the previously described species, the UreD-
UreF-urease apoprotein complexes were resistant to inactivation by NiClz. The bicarbonate
concentration dependence for UreD-UreF-urease apoenzyme activation was significantly
decreased compared to that of the urease and UreD-urease apoproteins. Western blot
(immtmoblot) analyses with polyclonal anti-urease and anti-UreD antibodies indicated
that UreD is masked in the UreD-UreF-urease complexes, presumably by UreF. I
propose that the binding of UreF modulates the UreD-urease apoprotein activation
properties by excluding nickel ions from binding to the active site until after formation of

the carbamylated lysine metallocenter ligand.

72
INTRODUCTION

Urease is a nickel-containing enzyme that catalyzes the hydrolysis of urea and
serves as a virulence factor in microorganisms that are associated with minary stone
formation, gastric ulceration, and other human health concerns (2, 7). Crystallogaphic
analysis of IGebsiella aerogenes urease revealed that the three structural subunits (encoded
by ureA, ureB, and ureC) associate into a trimer of trirners [(UreA-UreB-UreC)3] with each
UreC subunit containing a novel bi-nickel metallocenter (3). The two nickel ions, separated
by 3.5 A, are bridged by a carbamylated lysine residue. Ni-l is tricoordinate, bound by the
carbamate and two histidine ligands, whereas Ni-2 is pentacoordinate, including the
carbamate, two histidines, an aspartate, and a solvent molecule as ligands. The apoprotein,
formed in cells gown in the absence of nickel ion or generated in accessory gene deletion
mutants (5), is nearly identical to holoprotein in structure but lacks the metal ions and the
bormd carbon dioxide.

K aerogenes urease apoprotein, here termed Apo, can be partially activated in vitro
by incubation with nickel ions in bicarbonate-containing bufi‘ers (14). Carbon dioxide, in
equilibrium with bicarbonate, reversibly reacts with the active-site lysine residue to form the
requisite carbamate ligand. The concentration of carbon dioxide required for half-maximal
activation is 0.2%, or approximately sevenfold the atmospheric concentration of this gas.
Under optimal conditions, only 10-15% of the Apo is activated. The activation reaction
competes with nickel binding to noncarbamylated protein and with nonproductive nickel

binding to carbamylated protein (15). An additional complication is that several other metal

73
ions compete with nickel to form inactive metal-substituted species. Direct activation of

Apo likely does not occur to any significant extent in the cell (reviewed in reference 8).
Cellular activation of K aerogenes urease requires the participation of three
accessory proteins (UreD, UreF, and UreG) and is aided by a fourth (UreE) (5). These
peptides are encoded by genes located in the same cluster (ureDABCEFG’) as those
encoding the enzyme subunits (5, 9). On the basis of its ability to bind nickel ions (6), UreE
is suggested to play a role involving nickel donation or storage. In contrast, the other
accessory proteins are thought to function as complexes with the urease subunits. Both
ureD and ureF possess non-consensus ribosome binding sites and are expressed at very low
levels in the cell. Changing the ribosome binding site and initiation codon for ureD to yield
a more consensus-like sequence resulted in the synthesis of high levels of UreD, shown to
be present in a series of complexes containing the trimeric Apo [(UreA-UreB-UreC)3]
binding one, two, or three UreD peptides (12). For convenience, these UreD-mease
apoprotein complexes will be called D-Apo. Incubation of D-Apo with bicarbonate and
nickel ions leads to UreD dissociation and partial activation, accormting for approximately
30% of the available apoprotein (14). As found for Apo, nonproductive reactions compete
with the activation process. These reactions include nickel binding to the noncarbamylated
D-Apo, nickel binding improperly to the carbamylated species, and binding of other metal
ions to form inactive metal-substituted proteins (15). The formation of D-Apo is not
sumcient to account for activation of urease in the cell. Another series of Apo complexes
containing UreD, UreF, and UreG has been detected and is able to be activated (13). Since

all of these components are required for generating active urease in viva, the UreD-UreF-

74
UreG-Apo complexes (DFG-Apo) were suggested to serve as the key cellular urease

activation machinery. These complexes are present at minute levels in the cell and their
properties have not been well characterized (11). The specific roles for UreD, UreF, and
UreG in these complexes are unknown.

To better define the functions of the individual accessory proteins, we have
identified, purified, and characterized the properties of a new series of complexes comprised
of UreD, UreF, and the mease subunits. The UreD-UreF-Apo complexes (DF-Apo) have
activation properties that are distinct from those of Apo or D-Apo species and provide

insight into the role of UreF.

MATERIALS AND METHODS

Bacterial strains and plasmid constructions. All molecular biology methods
followed the general protocols outlined in Sambrook et al. ( 16).

Three constructs were generated in which ureF was overexpressed in a backgound
containing all or most of the other urease genes, with ureD also expressed at high levels in
two cases (Fig. 1). First, the 1.68-kbp SalI fragment of pKAUl7 (10) was bltmt ended with
Klenow fiagment and ligated to phosphorylated Ndel linker (5'-ACCATATG-3'). The
resulting product was digested with Ndel and Aer, and the 670-bp fragment was ligated to
the 5.7-kbp Ndel-Nhel fi'agnent of pET-l 1a (Novagen, Madison, WI), thus placing ureF

under the control of the T7 promoter. This construct, pETF, was transformed into

75

EcoRI A AflII
NruI
+

.euii: :
pKAUD2 * D A LB I C

 

 

 

 

 

 

 

pKAUD2rlF D THE] C

 

 

 

 

 

C

 

 

 

Figure 1. Schematic representations of several plasmids utilized in these studies. The
pKAUD2 or pKAU17 map shows several restriction sites used for plasmid constructions
as well as the gene arrangements in the plasmids. Transcription proceeds from left to
right. Note that pKAUD2 (12) is identical to pKAUl7 (10), except it is modified in the
ureD ribosome-binding site region and initiation codon in order to overexpress ureD

(shown as +). The pUC8 vector is indicated by (- ).

76
Escherichia coli BL21(DE3) (N ovagen, Inc.). The 740-bp XbaI-BamHI fragnent of pETF

was inserted into the blunt-ended EcaRl site of pKAUD2 (12) to form pKAUD2F+. A
ureG deletion derivative of this plasmid, pKAUD2F+AureG, was obtained by religation of
the bltmt-ended 8-kbp AvrII-Kanl fiagnent. The double deletion mutant,
pKAUD2F+AureDAureG, was constructed by digesting plasmid pKAUD2F+AureG with
NruI and AflII, filling in the AflII 5' overhang, and ligating the 7.1-kbp fragnent ends.

These plasmids were transformed into E. coli DH5a.

Culture conditions and cell disruption. E. coli DH5a(pKAUD2F+) or its
derivatives were gown at 30 or 37 °C to late stationary phase in Luria-Bertani (LB) broth
supplemented with 100 ug ampicillin per ml, harvested by centrifugation, and suspended in
PEDG (18 mM potassium phosphate [pH 7.4], 0.09 mM EDTA, 0.09 mM dithiothreitol,
10% glycerol) bufi‘er. Resuspended cells were disrupted by two to three passages through a
French pressrue cell at 18,000 Win2 (1 lb/in2 = 6.89 kPa), supplemented with 1 mM
phenylmethylsulfonyl fluoride, and separated into extracts and pellet fractions by

centrifugation at 100,000 x g for 45 min at 4 °C.

Purification or DF-Apo. Starting with E. coli DH5a(pKAUD2F+AureG) cell
extracts, the DF-Apo was purified by successive chromatography on DEAE-Sepharose,
Mono Q, and Superose 6 resins. The sample was applied to a DEAE-Sepharose column
(2.5 x 19 cm; Pharmacia Biotech, Piscataway, NJ) equilibrated in PEDG bufi‘er, and

proteins were eluted from the column by using a 400-ml linear salt gadient to 0.5 M KCl in

77
the same buffer. Fractions containing the DF-Apo were pooled, dialyzed against PEDG

bufier, and applied to a Mono Q HR 10/10 (Pharmacia Biotech) column equilibrated with
the same buffer. The proteins were eluted with a linear salt gadient to 1 M KCl in PEDG
buffer. DF-Apo eluted from the column at 0.4-0.6 M KCl. Fractions containing the
desired proteins were pooled, dialyzed against 18 mM phosphate [pH 7.4] bufl‘er
containing 0.09 mM EDTA, 0.09 mM dithiothreitol, and 0.1 M KCl. The protein pool
was concentrated in a Centriprep-IO or -30 (Amicon, Beverly, MA) to 4 mls and
chromatogaphed, via 2 runs, on a Superose 6 column (1.6 x 49 cm; Pharmacia Biotech)

equilibrated in the same buffer.

Polyacrylamide gel electrophoresis. Sodium dodecyl sulfate (SDS)—
polyacrylamide gel electrophoresis was canied out by using the buffers described by
Laemmli (4) and included 4.5% and 13.5% polyacrylamide stacking and rtmning gels.
Nondenattuing gels used the same bufi‘ers without detergent and consisted of 3% and 6%
polyacrylamide stacking and nmning gels. The gels were either stained with Coomassie
brilliant blue or electroblotted onto nitrocellulose, probed with anti-K aerogenes urease
antibodies (10) or anti-K aerogenes UreD antibodies (17), and visualized by using anti-
rabbit immtmoglobulin G-alkaline phosphatase conjugates ( l ). The band intensities of
Coomassie-stained gels were determined with an AMBIS (San Diego, CA) gel scanner. For
calculation of the ratios of UreD, UreF, and UreC, M, values of 29,300, 27,000, and 60,300

were used.

78

Urease activity assays. Urease activity was measured by quantitating the rate of
ammonia release from urea by formation of indophenol, which was monitored at 625 nm
(19). One unit of urease activity was defined as the amount of enzyme required to
hydrolyze 1 mole of urea per min at 37 °C. The standard assay buffer consisted of 25 mM
HEPES ( N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; pH 7.75), 0.5 mM EDTA,
and 50 mM urea. Protein concentrations were determined by a commercial assay (Bio-Rad,

Hercules, CA) with a bovine serum albumin standard.

Activation of urease apoproteins. Routine Apo, D-Apo, and DF-Apo activation
buffer consisted of 100 mM HEPES (pH 8.3), 150 mM NaCl, 100 mM NaHCO;, and 100
M NiCl2 (15). For specific experiments, the conditions were modified by varying the
bicarbonate or nickel ion concentrations, adding other metal ions to the activation bufl‘er, or

incubating samples in the presence of metal ions prior to addition of bicarbonate.

RESULTS AND DISCUSSION

Purification and characterization of UreD-UreF-urease apoprotein complexes.
E. coli DH5a(pKAUD2F+AureG) synthesized high levels of the urease subunits, UreD, and
UreF. When cells were gown in the absence of nickel ions these peptides were found to
copurify during DEAE-Sepharose, Mono Q, and Superose 6 chromatogaphies as illustrated
by denaturing polyacrylamide gel electrophoretic analysis of the resulting protein pools (F ig

2, lanes 2-5). On the basis of the staining intensities, the composition of the last pool was

79
calculated to be 0.86-0.89 UreD and 0.78-0.81 UreF per UreC. The broad elution profile

fiom the gel permeation column suggested that multiple DF-Apo species were present.
Results from nondenaturing polyacrylamide gel electrophoretic analyses were consistent
with the presence of three DF-Apo complexes when proteins were visualized by probing
with anti-urease irnmunoglobulin G (Fig. 3A, lane 3). The electrophoretic migation
positions of these complexes corresponded to those described previously for the D-Apo
complexes (12), as shown in Figure 3A, lane 2. Surprisingly, however, anti-UreD
antibodies failed to detect cross-reactive material associated with the DF-Apo bands (Fig.
3B, lane 3), whereas the D-Apo species were detected (Fig. 3B, lane 2). (When the same
fiactions were analyzed by Westem blot [irnmunoblot] analysis of a denattuing gel, UreD
was clearly detected by these antibodies [data not shown]). After incubation with nickel
ions in activation buffer, the three DF-Apo species collapsed to form a band that migated at
the position of urease and Apo (Fig. 3A, lane 4 and Fig. 3C), similar to the results reported
for D-Apo (12). Addition of bicarbonate alone had no efiect on the band migation. Two
bands (labelled X in Fig. 3) that migated more slowly than urease, but slightly faster than
the 1 UreD-Apo species were detected by Coomassie staining of the activated DF-Apo
species. These bands did not cross-react with anti-urease or anti-UreD antibodies. As
observed previously in cell extracts from E. coli DH5(pKAU17) (13), a slowly migating
band (labelled Y in Fig. 3) cross-reacted with anti-unease and anti-UreD antibodies. Two-
dimensional gels (data not shown) indicated that the predominant protein in this band is that
observed just below UreC in Figure 2. N-terrninal sequence analysis of this peptide

revealed a sequence consistent with that of GroEL (XXKDVKF). Pmified E. coli GroEL

80

 

Figure 2. Partial purification of the DF-Apo as analyzed by SDS/polyacrylamide gel
electrophoresis. Samples included: lane 1, molecular weight markers (phosphorylase b,
M, 97,400; bovine sermn albumin, M, 66,200; ovalbumin, Mr 45,000; carbonic anhydrase,
Mr 31,000; soybean trypsin inhibitor, Mr 21,000; and lysozyme, M, 14,400); lane 2, E. coli
DH5a(pKAUD2F+AureG) cell extracts; lane 3, DEAE-Sepharose pool; lane 4, Mono Q

pool; lane 5, Superose 6 pool.

81

FIG. 3. Native polyacrylanride gel electrophoretic comparison of the D-Apo and DF-Apo
samples. PM Western blot analysis of a nondenaturing polyacrylamide gel
visualized with anti- K. aerogenes urease antibodies. Samples included: lane 1, 10 pg
Apo; lane 2, 10 pg of D-Apo complexes; lane 3, 20 pg of DF-Apo; lane 4, 10 pg of DF-
Apo complexes incubated in activation buffer at 37 °C for 90 minutes. N Western
blot comparison using anti-UreD antibodies for samples as in panel A. _P_a_n_e&
Coomassie-stained native gel with the same protein sample as shown in lane 4 of panel A.
The migation positions for Apo and D-Apo containing 1, 2, and 3 UreD per trimeric
urease [(UreA-UreB-UreC)3] (12) are indicated. The band labelled Y contains GroEL

that crossreacts with anti-urease and anti-UreD antibodies.

 

 

83

was found to electrophorese at positions corresponding to these bands on native and
denaturing gels, and both antibodies were observed to cross-react with authentic GroEL. I
conclude that GroEL is present at substoichiometric levels in our preparations of D-Apo and
DF-Apo.

From the results described above, I conclude that the DF-Apo species represent the
trimeric Apo [(UreA-UreB-UreC)3] with 1, 2, or 3 each of UreD and UreF peptides bound.
On the basis of Western blot analyses, 1 suggest that the UreF peptides mask the UreD
peptides; i.e., anti-UreD antibodies fail to detect the UreD that is present in the DF-Apo
although the peptide is recogrized in D-Apo. Although DF-Apo and D-Apo complexes
appear to migate identically when examined by native gel electrophoresis, slight changes in
size are detected when these samples are examined by gel permeation chromatogaphy. It is
likely that the DF-Apo complexes were unknowingly observed before. In prior studies that
sought to examine the requirements for D—Apo formation, deletion mutants in ureE, ureF,
or ureG were studied by immunological methods using E. coli cells containing the K
aerogenes urease gene cluster (13). Although the D-Apo complexes in AureF mutant cells
comi gated with complexes from control and AureE or AureG mutant cells during
electrophoresis, the absence of UreF led to an apparent diminishment in gel chromatogaphy
size that was attributed to a conformational change induced by UreF. The present findings

allow reinterpretation of the prior results: the control and AureE or AureG mutants actually

formed DF-Apo and only the AureF mutant cells formed D-Apo.
To examine whether UreD was needed for UreF to form a complex with the mease

subunits, pKAUD2F+AureDAureG was constructed. Cells containing this plasmid were

84
disrupted, and extracts were chromatogaphed on DEAE-Sepharose and Mono Q columns.

The urease subunits eluted from these resins as noncomplexed species and UreF appeared to
be almost entirely insoluble. Furthermore, when ureF was expressed separately fiom the
genes encoding the urease subunits, the gene product was exclusively located in the

insoluble fraction (data not shown).

Activation properties of DF-Apo. When standard Apo activation conditions were
used, the DF-Apo pool was activated to a specific activity value of 800 :t 100 U/mg of total
protein (e.g., see closed triangles in Fig. 4 and Fig. 5A). This level of activation was very
similar to that observed for D-Apo and about twice that obtained for Apo (14, 15). For
comparison, native urease isolated from K aerogenes possesses a specific activity of ~2,500
U/mg of protein (1 8). The rate of DF-Apo activation, however, was sigrificantly slower
than that observed with either of the other apoproteins when assessed under similar
conditions. For example, 60-90 min was required for half-maximal activation of DF-Apo,
compared with 15-20 min for the Apo and D-Apo species (14).

The nickel and bicarbonate concentration dependencies were sigrificantly altered
for the new species compared to the corresponding properties for Apo and D—Apo. As
shown in Fig. 4, the nickel ion concentration exhibited a marked effect on the DF-Apo
activation rate, with increasing rates observed to 500 pM and slight inhibition observed at 1
mM NiCl2. In contrast, the Apo and D-Apo species exhibit maximal activation rates at ~60
pM NiCl2 and are inhibited by nickel ion concentrations above 300 pM Ni (14). For DF-

Apo, half-maximal levels of activity were achieved at ~5 mM added bicarbonate when

85

900 -

800 ‘

 

700 -

600 1

500 -

400 -

300 -

Specific Activity (U/mg)

200 —

100 -

 

/

0 I I I I I I I I
0 20 40 60 80 100 120 140 160

Time (min)

Figure 4. Nickel ion concentration dependence of activation for the DF-Apo. The
purified sample (0.1 mg/ml) was incubated at 37 °C in 100 mM HEPES (pH 8.3) bufl'er

containing 150 mM NaCl, 100 mM NaHCO;, and 25 (O), 50(I), 100(A), 500(0), or 1000
(El) pM NiCl2. Aliquots were removed at the indicated times and the samples were assayed

for urease activity.

86
using 100 pM NiC12 (Fig. 5A) or at ~250 pM sodium bicarbonate when 500 pM NiCl2 (Fig.

5B) was used. These results contrast those obtained with Apo and D-Apo species, which
exhibit half-maximal activation in the presence of approximately 10 mM bicarbonate.

As another measure to compare activation properties of DF-Apo versus Apo and D-
Apo species, the ability for various metal ions to inhibit the activation process was studied.
Prior incubation of DF-Apo with nickel ions in the absence of added bicarbonate led to only
a slight reduction in its ability to be activated (Fig. 6). Prior work demonstrated that these
same conditions geatly reduced the activation competence of Apo and D-Apo (15). On the
other hand, DF-Apo was almost completely inhibited by prior incubation with 100 pM zinc,
cobalt, or copper ions (Fig. 6), similar to the behavior of Apo and D-Apo samples (15). As
observed with the previously characterized species, inhibition of DF-Apo resulted when
activation was carried out with buffers containing 100 or 500 pM NiCl2 and varied
concentrations of zinc ions (Fig. 7). For example, only about 50% of the urease in the pool
was activated with 100 pM NiC12 in the presence of ~3.3 pM zinc ions. In the presence of
500 pM NiCl2, a slightly higher concentration of zinc ion (4.3 M was required to inhibit

activation by 50%.

Function of UreF. The enhanced activation properties of DF-Apo over D-Apo and
Apo (i.e., an increased resistance to nickel ion inhibition and a decreased bicarbonate
concentration dependence) provide compelling evidence that this novel complex is
biologically relevant. Although DF-Apo may not exist to any sigrificant extent in the cell,

it may occur as an intermediate to formation of the DFG-Apo species and it appears to

87

FIG. 5. Bicarbonate ion concentration dependence of activation for the DF-Apo. The

sample (0.1 mg/ml) was incubated at 37 °C in 100 mM HEPES (pH 8.3) buffer containing
150 mM NaCl, 100 (upper panel) or 500 pM (lower panel) NiCl2, and the following
concentrations of NaHCO3: 0(0), 0.1 (A), 0.25 (CI), 1 (V), 2 (V), 5 (O), 10 (O), 25

(0). 50 (I), 75 (O ), or 100 (A) mM added NaHCO3.

88

 

 

 

 

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89

provide insight into the role of UreF. I propose that UreF modulates the activation process
primarily by eliminating the binding of nickel ions to noncarbamylated protein. This
change geatly facilitates formation of the carbamylated protein, which in turn results in a
lowered bicarbonate requirement. As in the Apo and D-Apo species (15), however, the
carbamylated DF-Apo protein is likely to bind nickel ions to yield two states, only one of
which is active. Thus, in vitro activation of DF-Apo yields a final specific activity that falls
far short of that observed (~2,500 U/mg) in fully active enzyme (1 8). The ratio of active to
inactive nickel-containing carbamylated protein arising from DF-Apo is likely to be similar
to that generated from D-Apo, based on their similar final specific activities of ~800 U/mg
total protein. In addition to eliminating the interaction of nickel ions with noncarbamylated
urease apoprotein, an added consequence of forming the DF-Apo is to reduce the overall
activation rate by approximately fivefold. Additional studies are needed to elucidate how
UreF acts to preclude nickel ion from binding to the noncarbamylated apoenzyme complex,
and why zinc, cobalt, and copper ions do not appear to be similarly excluded. Furthermore,
it will be important to purify the DFG-Apo species and characterize how its activation

properties compare to those of Apo, D-Apo, and now DF—Apo.

90

700 -

 

600 4

 

500 -

400 -

300 -

Specific Activity (U/mg)

200 -

100 -

 

 

 

0 50 1 00 1 50 200

Time (min)

Figure 6. Effect of incubation of DF-Apo with metal ions prior to activation by NiC12 and

bicarbonate. The sample (0.5 mg/ml) was incubated at 37 °C in buffer containing 100 mM
HEPES (pH 8.3) and 150 mM NaCl with no metal (O) or with 100 pM NiCl2 (I), Zan
(0), C002 (A), or Cqu (El). After 80 minutes an aliquot of each mixture was activated in
100 mM HEPES (pH 8.3) buffer containing 150 mM NaCl, 100 pM NiCl2, and 100 mM

NaHCO3, and samples were assayed for urease at the indicated timepoints.

91

Percent specific activity

 

 

 

1 10 100
Zn ion (pM)

Figure 7. Effect of zinc ion concentration on DF-Apo activation. The sample (0.1 mg/ml)
was incubated in activation buffer containing 100 (I) or 500 (O) M NiC12 and the
indicated concentrations ZnCl2. The percent specific activity, based on a sample containing

no zinc ions, was determined after 4 hours incubation.

10.

ll.

92
REFERENCES

Blake, M. S., Johnston, K. H., Russel-Jones, G. L. & Gotschlich, E. C. (1984).
A rapid, sensitive method for detection of alkaline phosphatase-conjugated
anti-antibody on western blots. Analytical Biochemistry 136, 175-179.

Hausinger, R. P. (1993). Urease. In Biochemistry of Nickel, pp. 23-57. New
York: Plenum Publishing Corp.

Jabri, E., Carr, M. B., Hausinger, R. P. & Karplus, P. A. (1995). The crystal
structure of urease from Klebsiella aerogenes. Science 268, 998-1004.

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature (London) 227, 680-685.

Lee, M. H., Mulrooney, S. B., Renner, M. J., Markowicz, Y. & Hausinger, R.
P. (1992). Sequence of ureD and demonstration that four accessory genes (ureD,

ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis. Journal
of Bacteriology 174, 4324-4330.

Lee, M. H., Pankratz, H. S., Wang, S., Scott, R. A., Finnegan, M. G.,
Johnson, M. K., Ippolito, J. A., Christianson, D. W. & Hausinger, R. P.
(1993 ). Purification and characterization of Klebsiella aerogenes UreE protein: a
nickel-binding protein that functions in urease metallocenter assembly. Protein
Science 2, 1042-1052.

Mobley, H. L. T., Island, M. D. & Hausinger, R. P. (1995). Molecular biology
of microbial ureases. Microbiological Reviews 59, 451-480.

Moncrief, M. B. C. & Hausinger, R. P. (1996). Nickel incorporation into
urease. In Mechanisms of Metallocenter Assembly, pp. 151-171. Edited by R. P.
Hausinger, G. L. Eichhom & L. G. Marzilli. New York: VCH Publishers.

Mulrooney, S. B. & Hausinger, R. P. (1990). Sequence of the Klebsiella
aerogenes urease genes and evidence for accessory proteins facilitating nickel
incorporation. Journal afBacterialagy 172, 5837-5843.

Mulrooney, S. B., Pankratz, H. S. & Hausinger, R. P. (1989). Regulation of
gene expression and cellular localization of cloned Klebsiella aerogenes (K.
pneumaniae) urease. Journal of General Microbiology 135, 1769-1776.

Park, I.-S. (1994). Thesis. East Lansing, MI: Michigan State University.

12.

13.

14.

15.

16.

17.

18.

19.

93

Park, I.-S., Carr, M. B. & Hausinger, R. P. (1994). In vitro activation of urease
apoprotein and role of UreD as a chaperone required for nickel metallocenter
assembly. Proceedings of the National Academy of Sciences 91, 3233-3237.

Park, I.-S. & Hausinger, R. P. (1995). Evidence for the presence of urease
apoprotein complexes containing UreD, UreF, and UreG in cells that are
competent for in vivo enzyme activation. Journal of Bacteriology 177, 1947-
1951.

Park, I.-S. & Hausinger, R. P. (1995). Requirement of carbon dioxide for in
vitro assembly of the urease nickel metallocenter. Science 267 , 1156-1158.

Park, I.-S. & Hausinger, R. P. (1996). Metal ion interactions with urease and
UreD-urease apoproteins. Biochemistry 35, 5345-5352.

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular cloning: a
laboratory manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory.

Taha, T. & Hausinger, R. P. (1996). Unpublished observations.
Todd, M. J. & Hausinger, R. P. (1987). Purification and characterization of the
nickel-containing multicomponent urease from Klebsiella aerogenes. Journal of

Biological Chemistry 262, 5963-5967.

Weatherburn, M. W. (1967). Phenol-hypochlorite reaction for determination of
ammonia. Analytical Chemistry 39, 971-974.

CHAPTERS

CHARACTERIZATION OF UREG, IDENTIFICATION OF A URED-UREF-UREG
COMPLEX, AND EVIDENCE THAT A FUNCTIONAL NUCLEOTIDE-BINDING
SITE IS REQUIRED FOR IN VIVO METALLOCENTER ASSEMBLY OF

UREASE

94

95
ABSTRACT

Previous studies have indicated that urease metallocenter assembly in Klebsiella
aerogenes requires the presence of several accessory proteins (UreD, UreE, UreF, UreG) in
addition to the urease structural subunits. UreG was isolated by using a novel purification
procedure that included sequential ion exchange chromatogaphic steps that used phosphate
buffers containing or lacking 20 percent glycerol. The protein was shown to be a monomer
of M, = 21,814 i 20 by using a combination of gel filtration chromatogaphy and mass
spectrometry. Although it contains a P-loop motif typically found in nucleotide-binding
proteins, UreG did not bind or hydrolyze ATP or GTP and it exhibited no amnity for ATP-
and GTP-linked agarose resins. Site-directed mutagenesis of ureG allowed the production
of altered UreG proteins at the P-loop motif. Substitution of alanine at positions Lys-20 or
Thr-21 resulted in the production of inactive urease in cells gown in the presence of nickel,
indicating the importance of an intact P-loop for UreG function in vivo. These mutant cells
were unable to synthesize the UreD-UreF-UreG-urease apoprotein complexes, thought to be
the key activation complexes in the cell. A novel complex containing UreD, UreF, and
UreG (termed DFG complex) was detected in cells carrying the urease gene cluster with
deletions of ureE and the urease structural genes. The DF G complex was found to be in the
insoluble fiaction upon cell lysis, but was solubilized by addition of Triton X-100.
Following DEAE-Sepharose chromatogaphy and removal of detergent, the DF G complex
bound to an ATP-linked agarose resin. In cells containing a UreG P-loop variant, the DF G

complex was formed but it did not bind to the nucleotide-linked resin. These results

96
suggest that the UreG P-loop motif is essential for nucleotide binding in the DFG complex

and that nucleotide hydrolysis may be required for in viva metallocenter assembly.

97
INTRODUCTION

In viva activation of Klebsiella aerogenes urease involves the participation of four
accessory proteins: UreD, UreE, UreF, and UreG (1). Although the precise ftmction of
these proteins in urease nickel metallocenter assembly has not been established, UreE has
been shown to be a nickel-binding protein (2), and a series of UreD-urease (D-Apo) (3),
UreD-UreF-urease (DF-Apo) (4, Chapter 4), and UreD-UreF-UreG-urease (DFG-Apo) (5)
apoprotein complexes have been identified. The auxiliary proteins within these complexes
alter the activation properties of the urease apoprotein (Apo) and allow the cell to obtain
fully active enzyme. Apo activation has been studied in vitro and shown to require the
presence of carbon dioxide (6). Structural studies have demonstrated that the essential C02
is used to form a carbamylated lysine at the active site which functions as a bridging ligand
to the requisite nickel ions (7).

0f the four urease accessory proteins, UreG is the most highly conserved (Chapter
1, see section HF) and the only one of this goup that exhibits clear sequence homology to
other proteins. For example, UreG contains a P-loop motif that is typically found in ATP-
and GTP-binding proteins (8) where it functions in nucleotide binding. The presence of this
putative nucleotide-binding site in UreG might be related to the in viva energy requirement
for urease activation (9). Besides the limited similarity to the P-loop motif in various
proteins, the entire UreG protein from K. aerogenes is approximately 25% identical to the
sequence of the Escherichia coli hypB gene product (10). This gene is part of the

hydrogenase pleiotropic operon required for activation of the three nickel-containing

98

hydrogenases found in E. coli. HypB plays a role in nickel-ion processing based on the
demonstration that mutations in hypB can be complemented by addition of high levels of
nickel ion to the gowth medium (1 1). E. coli HypB has been purified and found to bind
and hydrolyze GTP (12). These findings raise the possibilities that UreG may bind
nucleotides and catalyze nucleotide hydrolysis as part of its role in facilitating urease
metallocenter assembly.

Here, I describe the purification and characterization of UreG and I provide
evidence from site-directed mutagenesis studies that the P-loop motif is needed for UreG to
function in viva in metallocenter assembly. In addition, I demonstrate the presence of a
complex (termed DFG) containing UreD, UreF, and UreG. Partially purified DF G complex
is shown to bind to a nucleotide-linked resin, whereas DFG complex obtained from a P-
loop variant lacks the ability to bind to this resin. These studies highlight the importance of

the P-loop motif within UreG and identify a novel complex of the urease accessory proteins.

MATERIALS AND METHODS

Bacterial strains and plasmid construction. All molecular biology methods followed
the general methods outlined in Sambrook et al. (13). All plasmids were transformed

into E. coli DHSa.

To test the necessity for the P-loop motif in UreG, two mutants were created in
which Lys-20 was changed to Ala (K20A) or Thr-21 was changed to Ala (T21A). A 1.5-

kb SalI-Kpnl fragnent of pKAU17 (14) was subcloned into M13mp18 and mutagenized

99
by the method of Kunkel et al. (15). Uracil-containing single-stranded template DNA

was prepared from E. coli CJ236 [dutl ungl thi-1 relAl/pCJ 105(cam'F’)]. By using
oligonucleotide primers 5’-TCGGCTCCGGT@AACCGCTCTGC-3’ or 5’-
GCTCCGGTAAAQCCGCTCTGCTGJ’ (the underlined bases represent changes from
the wild-type sequence) , the phage DNA was mutagenized and the mutant phage were
isolated in E. coli MV1193 (A[lac-praAB] rpsL thi endA spcB15 hst4 A[srl-
recA]306::Tn10[tet’] F’ [traD36 praAB+ laclq lacZAMlS). Mutants were identified by
sequence analysis using Sequenase 2.0 (United States Biochemical) and the single-strand
DNA sequence method of Sanger et al. (16). The 840-bp AvrII-KpnI fragnent was
subcloned back into pKAUl7 to generate pKAU17K20A or pKAU17T21A and the
sequence was confirmed by double-strand DNA sequencing methods. The 2.4-kb
BamHI-Kpnl fragnents of pKAU17K20A and pKAU17T21A were also subcloned into
the 5.7-kb BamHI-Kpnl fragnent of pKAUD2, containing a mutated ureD promoter
region to allow overexpression of this gene (3), to generate pKAUD2K20A or

pKAUD2T2 l A.

Plasmids were also constructed with ureG or a mutated ureG gene for study of the
DFG complex. The construct pKAUD2F+AureAAureBAureCAureE containing only
ureD, ureF, and ureG was created by taking the 5.4-kb AflII-Blnl fragnent of
pKAUD2F+ (17), blunt-ending with Klenow fragnent, and religating the ends. The
pKAUD2F+T21AAureA AureBAureCAureE plasmid was constructed by ligating a blunt-
ended 8.2-kb EcoRl fragnent of pKAUD2T21A to a blunt-ended 740-bp XbaI-BamHl

pETF fragnent (4) containing the overexpressed ureF gene. This plasmid,

100
pKAUD2F+T21A, was digested with AflII and BlnI, the 5.4-kb fragnent was blunt ended

with Klenow, and the ends were ligated together. For routine purification of UreG, I
made use of plasmid pKAUG-l containing only ureG (17). This plasmid was constructed
by isolating the 4.1 kb KspI-EcaRl from pKAUD2 (3), blunt-ending with Klenow, and

ligating the ends together.

Culture Conditions. All cultures were gown at either 30 °C or 37 0C to late stationary
phase in 3 liters of Luria-Bertani (LB) broth supplemented with 100 pg ampicillin per ml.
E. coli DHS cells containing plasmids pKAUG-l, pKAU17K20A, pKAUl7T21A,
pKAUD2K20A, or pKAUD2T21A were harvested by centrifugation and resuspended in
30 mls PEB (20 mM phosphate [pH 7.4], 1 mM EDTA, 1 mM 2-mercaptoethanol) buffer.
E. coli DHS cells containing plasmids pKAUD2F+AureAAureBAureCAureE or
pKAUD2F +T21AAureAAureBAureCAureE were harvested by centrifugation and
resuspended in 30 mls of HMDG (25 mM HEPES [pH 7.4], 5 mM MgC12, 0.5 mM

dithiothreitol, 10 percent glycerol) buffer.

Purification of UreG. E. coli DHSor cells carrying plasmid pKAUG-l were resuspended
in PEB buffer, supplemented with 1 mM phenylmethylsulfonyl fluoride, and disrupted by
passage through a French pressure cell (American Instrument Co., Silver Spring, MD) at
18,000 lb/in2 (l lb/in2 = 6.89 kPa). Cell extracts, obtained after centrifugation at 100,000

x g for 45 minutes at 4 °C, were applied to a DEAE-Sepharose column (2.5 x 19 cm;

101
Pharmacia Biotech, Piscataway, NJ) that had been equilibrated with PEB, and proteins

were eluted by using a 400-ml linear salt gadient to 0.5 M KCl in the same buffer.
Fractions containing UreG (eluting at approximately 0.1-0.2 M KCl) were pooled,
dialyzed against PEB buffer containing 20 % glycerol, and applied to a Mono Q HR
10/10 (Pharmacia Biotech) column equilibrated with the same buffer containing 20%
glycerol. F low-through fi'actions containing UreG were pooled, dialyzed against PEB
buffer to remove the glycerol, and applied to the re-equilibrated Mono Q column in PEB
buffer. A linear salt gadient to a concentration of l M KCl in PEB was used to elute
UreG at approximately 0.1-0.4 M KC1. Samples containing UreG were pooled,
concentrated, dialyzed against 25 mM HEPES buffer (pH 7.4) containing 1 mM EDTA, 1
mM 2-mercaptoethanol, and 0.1 M KCl, and subjected to Superose l2 chromatogaphy in
this same eluent. For subunit molecular weight determination, matrix-assisted laser
desorption ionization time-of-flight mass spectrometry was used. A 0.6 pg sample of
purified UreG was loaded onto Zetabind nylon membrane, dried, washed with water, and
mixed with one pl of sinapinic acid (18). The sample was run on a Vestec LaserTec
Research time-of-flight mass spectrometer (V estec, Houston, TX) equipped with a

nitrogen laser (model VSL-337ND, Laser Science, Newton, MA).

Partial purification of the DFG complex. Three liters of cells of E. coli DHSor carrying

plasmids pKAUD2F+AureAAureBAureCAureE or pKAUD2F+T21AAureAAureBAureC-

AureE were resuspended in HMDG buffer, supplemented with 1 mM phenyhnethyl-

sulfonyl fluoride, and disrupted by passage through a French pressure cell at 18,000

102
lb/inz. The sample was centrifuged at 27,000 x g for 45 minutes at 4 °C and the

supernatant was removed. The pellet was washed with 20 ml of HMDG buffer,
recentrifuged as stated above, and the supernatant was removed. The pellet was
suspended in 20 ml HMDG buffer containing 0.5% (w/v) Triton X-100 or Tween 20 and
incubated at 4 °C for 17 h. After centrifirgation at 27,000 x g for 45 min, the supernatant
fiaction containing the DFG complex was removed. The solubilized protein sample was
applied to a DEAE-Sepharose column (2.5 x 19 cm) that had been equilibrated with
HMDG buffer containing detergent, and the proteins were eluted by using a 400-ml linear
salt gadient to 0.5 M KCl in the same buffer. Fractions containing DF G were pooled,
dialyzed against HMDG buffer with 0.5% detergent, and added to a column of Extracti-
Gel D Detergent Removing Gel (1.7 x 7 cm; Pierce, Rockford, IL). The flow-through
fractions containing DFG complex were pooled and added to ribose hydroxyl-linked GDP-,
ribose hydroxyl-linked GTP-, or N-6-linked ATP-agarose resins (Signa Chemical, St.
Louis, MO) that had been equilibrated in HMDG buffer. Columns were washed with

HMDG buffer and proteins were eluted with 10 mM ATP or 1 M KCl in HMDG buffer.

Preparation of polyclonal antibodies against UreG. Antibodies were generated in a
white, female, New Zealand rabbit by injecting 200 pl (2.5 mg/ml) of purified native
UreG in phosphate-buffered saline that was emulsified with the same volume of TiterMax
adjuvant (V axcel, Norcross, GA). The rabbit was boosted after 23 days, and after 97 days

the immunoglobulin G fraction was purified from the serum using the E-Z-Sep kit

103
(Pharmacia Biotech). Antibodies were titrated by using standard dot blot (l9) and

immunoblotting techniques (20).

Polyacrylamide gel electrophoresis. Sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis was carried out by using the buffers of Laemmli (21) and included 4.5%
and 12.5% polyacrylamide stacking and running gels. Nondenaturing gels used the same
buffers without SDS and consisted of 3% and 6% polyacrylamide stacking and rtmning
gels. Denaturing gels were stained with Coomassie brilliant blue, while nondenaturing
gels were electroblotted onto nitrocellulose, probed with either polyclonal anti-K.
aerogenes urease antibodies (14) or anti-K aerogenes UreG antibodies, and visualized
using goat anti-rabbit immunoglobulin G-alkaline phosphatase conjugates (Signa
Chemical, St. Louis, MO). When testing for the presence of biotin, blots were probed
and detected with ExtrAvidin-alkaline phosphatase (Signa Chemical). Cytochrome c-
biotin labeled protein (Signa Chemical) was used as a positive control. The band
intensities of Coomassie-stained gels were determined with an AMBIS (San Diego, CA) gel
scanner. For calculation of the ratios of UreD, UreF, and UreG, M, values of 29,300,

27,000, and 21,800 were used.

Urease activity assays. Urease activity was measured by quantitating the rate of
ammonia release from urea by formation of indophenol, which was monitored at 625 nm

(22). One unit of urease activity was defined as the amount of enzyme required to

104
hydrolyze 1 pmole of urea per minute at 37 °C. The standard assay buffer consisted of 25

mM HEPES (pH 7.75), 0.5 mM EDTA, and 50 mM urea. Protein concentrations were
determined by a commercial assay (Bio-Rad, Hercules, CA) with bovine serum albumin

standard.

Equilibrium dialysis determination of nucleotide binding. Nucleotide binding was
determined by equilibrium dialysis using 50 mM sodium phosphate (pH 7.2) buffer
containing 1 mM Mng, 0.5% NaCl, and the desired nucleotide or 50 mM Tris (pH 7.5),
100 mM NaCl, 5 mM Mng, 0.09 mM dithiothreitol, and 0.09 mM EDTA and the
desired nucleotide. ['4C]ATP (44 mCi/mmol; ICN Pharmaceuticals, Costa Mesa, CA) or
[MCJGTP (13.5 Ci/mmol; ICN Pharmaceuticals) was diluted with various concentrations
of unlabeled ATP or GTP, respectively. The protein concentration was 2 pM or 6 pM.
Free ligand equilibrium was attained in an equilibrium dialyzer (Hoeffer Scientific, San
Francisco, CA) using Spectrapor 2 dialysis membranes at 4 °C for approximately 14
hours. Samples of 100 pl were removed from each compartment and radioactivity was

measured in a liquid scintillation counter.

Chromatographic measurement of nucleotide hydrolysis. The rates of hydrolysis of
ATP and GTP were analyzed by using a C13 reverse-phase HR 5/5 column (Pharmacia
Biotech) equilibrated with buffer containing 19% acetonitrile, 30 mM potassium

phosphate, and 10 mM tetrabutylammonium phosphate (pH 2.65) (23) or analyzed by a

105
Mono Q HR 10/10 column equilibrated with 20 mM phosphate (pH 7.3). For reverse-

phase chromatogaphy, 0.033 pM of UreG, 0.6 mM ATP, and 10 mM MgC12 in 0.1 M
potassium phosphate buffer (pH 7.3) were incubated at room temperature for 1-13 hours.
Reactions were quenched with two volumes of column equilibration buffer and 1/10 of
the sample was chromatogaphed. GTP hydrolysis was measured using Mono Q
chromatogaphy. [3H]GT'P was diluted with unlabeled GTP to a final concentration of 5
pM and incubated at room temperature with 1.2 pM of UreG, 10 mM MgCl2, and 50 mM
Tris (pH 8.0). At various time intervals, samples were removed and chromatogaphed on
the Mono Q column. Radioactivity in the resulting fiactions was detected using a liquid

scintillation counter.

Carbonic anhydrase assay. Carbonic anhydrase activity was tested by monitoring the

hydrolysis of p-nitrophenyl acetate at 400 nm and 25 °C (24).

RESULTS

UreG purification and characterization. UreG was purified fiom E. coli cells
containing pKAUG-l by using a combination of DEAE-Sepharose, Mono Q, and
Superose 12 chromatogaphies (Fig. 1). From a one liter culture about 10-15 mg of UreG
was recovered. A novel step in the purification process involved sequential Mono Q
chromatogaphy of the sample in the presence and then absence of 20% glycerol in

phosphate buffer. UreG exhibited little affinity for the Mono Q column in the presence of

106
glycerol (Fig. 1, lane 4), but bound tightly to the resin when the glycerol was removed by

dialysis. This behavior was only observed when using phosphate buffers (i.e., the protein
was retained by the column in HEPES buffer containing 20% glycerol). UreG also
tended to smear when chromatogaphed on the Mono Q column in the absence of
glycerol. For most applications, the protein was essentially homogenous after the second
Mono Q chromatogaphy step (Fig. 1, lane 5); however, a Superose 12 column was added
as an additional purification step to prepare the sample for use in antibody production.
The estimated size of native UreG based on the gel filtration chromatogaphy results was
approximately 19.1 kDa. A more precise size estimate of UreG was determined by using
matrix-assisted laser desorption ionization time-of-flight electrospray-mass spectrometry.
UreG was shown to possess an M, of 21,814 i 20 which agees well with the expected
size (M, = 21,812) for protein lacking the N- terminal methionine based on the translated
DNA sequence (25).

To begin to characterize the fimction of UreG, I examined the properties of the
purified protein: its ability to bind or hydrolyze nucleotides, its carbonic anhydrase
activity, and its biotin content. The presence of the P-loop motif in the UreG sequence is
consistent with a role in nucleotide binding; however, no detectable ATP- or GTP-
binding was observed using equilibrium dialysis methods, and no nucleotide hydrolysis
was detected using two chromatogaphic procedures. Since high levels of CO2 are known
to be required for carbamylation of Lys-217 in the urease apoprotein (6) in order to form
the binuclear active site (7), I tested whether UreG possessed carbonic anhydrase activity

(to provide CO2 from bicarbonate) or whether it contained a cofactor that could function

107

 

w an“ -UreG

 

Figure 1. Purification of UreG as monitored by SDS/polyacrylamide gel electrophoresis.
Samples included: lane 1, molecular weight markers (phosphorylase b, M, 97,400;
bovine serum albumin, M, 66,200; ovalburnin, M, 45,000; carbonic anhydrase, M, 31,000;
soybean trypsin inhibitor, M, 21,000; and lysozyme, M, 14,400); lane 2, E. coli(pKAUG—
1) cell extracts; lane 3, DEAE-Sepharose pool; lane 4, first Mono Q pool using bufl‘er

containing glycerol; lane 5, second Mono Q pool using glycerol-free buffer.

108
to deliver CO2. No carbonic anhydrase activity was detected, and UreG did not contain

biotin when analyzed by Western blot (immunoblot) analysis using avidin-linked alkaline
phosphatase (data not shown). The latter result was consistent with mass spectrometric

size analyses (see above).

Site-directed mutagenesis of UreG. To further assess the importance of the highly
conserved P-loop motif in UreG, I used site-directed mutagenesis methods to prepare
constructs that formed altered UreG species. Since the Lys and Thr residues in the motif
are essential for nucleotide binding in a variety of proteins (8), Lys-20 and Thr-21 of the
UreG P-loop motif were changed independently to Ala to generate K20A and T21A
forms of this protein. The two mutated genes were subcloned back into pKAU17 and
pKAUD2. The UreG P-loop variants did not affect formation of the D-Apo or DF-Apo
complexes, but they prevented formation of DFG-Apo when E. coli cells containing
pKAUD2K20A or pKAUD2T21A were gown in the absence of nickel (Fig. 2).
Moreover, the P-loop specific alterations to pKAUl7 and pKAUD2 resulted in the
complete loss of urease activity when cells were gown in LB containing nickel (data not

shown).

Partial purification and characterization of the DFG complex. Since direct
purification and characterization of the DFG-Apo complexes (thought to be the key

activation complexes in the cell) remain an intractable problem, I examined the feasibility

109

GroEL-
DFG-Apo<

ZD-Apo- W
1 D-Apo-

Figure 2. Effects of alterations to the UreG P-loop on formation of urease apoprotein
complexes. Cell extracts were analyzed on a nondenaturing polyacrylamide gel and
probed with anti-K. aerogenes urease antibodies. Lanes: 1, 40 pg of cell extracts from E.
coli(pKAUD2); 2, 40 pg of cell extracts from E. coli(pKAUD2K20A); 3, 40 pg of cell
extracts from E. coli(pKAUDT21A). The positions of the co-migating D-Apo and DF-
Apo complexes (containing one, two, or three UreD or UreD and UreF molecules per
urease apoprotein trimer; 3, 4) and the DFG-Apo complexes (5) are indicated. The anti-

urease antibodies cross-react with GroEL that is present in these extracts (4).

 

110

97.4-
66.2-

45-

3 1_ '7

 

21-

Figure 3. Analysis of the UreD-UreF-UreG complex bound to an ATP-linked agarose
resin as analyzed by SDS/polyacrylamide gel electrophoresis. Samples included: lane 1,
molecular weight markers; lane 2, Extracti-Gel DFG pool; lane 3, sample eluted from

ATP-linked agarose resin by l M KCl in HMDG buffer.

111

of first characterizing the heterotrimeric accessory protein complex. The DFG complex
was partially purified from E. coli cells containing pKAUD2F+AureAAureBAureCAureE
using detergent solubilization, DEAE-Sepharose, and Extracti-Gel D chromatogaphy.
Based on staining intensities, the DFG complex consisted of 0.847-0.848 UreD and
0818-0819 UreF per UreG. The resulting DFG pool was added to various nucleotide-
linked agarose resins to test for nucleotide-binding capabilities. The DFG complex did
not bind to GDP- or GTP-linked agarose resins, but a variable portion of it was bound to
an ATP-linked agarose column. The bound complex eluted from the column upon
addition of HMDG buffer containing 1 M KCl (Fig. 3) or 10 mM ATP (data not shown).
If samples were not frrst chromatogaphed on a DEAE-Sepharose column, the DFG
complex did not bind to the ATP-linked agarose resin consistent with the removal of a
competing ligand during ion-exchange chromatogaphy. Interaction with the nucleotide-
resin also required the removal of detergent. The detergent-flee complex was rather
unstable and UreD precipitated out of solution in less than one week.

E. coli cells containing pKAUD2F+T2lAAureAAureBAureCAureE were
examined to determine whether the DFG complex was able to form and to determine
whether it had similar properties when the UreG was altered at the P-loop motif. The
cells were cultured and proteins were purified as described above. In cells containing the
UreG P-loop variant, the DFG complex was formed (data not shown); however, the
complex did not bind to the ATP-linked agarose resin as observed with the wild-type

DFG (data not shown).

1 12
DISCUSSION

UreG purification and partial characterization. The ureG gene is expressed at high
levels in K. aerogenes. Since this protein is required for functional nickel insertion into
the urease active site and contains a sequence suggestive of frmction (i.e., a P-loop motif)
its purification and characterization was a logical step in examining metallocenter
assembly. UreG was purified by using a novel pmification procedure in which addition
and then removal of 20 percent glycerol fi'om a phosphate buffer resulted in geatly
altered ion-exchange chromatogaphic properties of the protein. It is unclear how
glycerol affects the chromatogaphic behavior of UreG; however, this type of result is not
unprecedented. For example, the presence of solvent-excluding reagents like
polyethylene glycol was found to affect the self-association of Rubisco activase so that
the molecular mass was two- to four-fold higher in the presence of this reagent, and the
enzyme exhibited higher ATPase and Rubisco activating properties in addition to
possessing increased affinity for ATP and Rubisco (26). The structural changes in UreG
appear to depend on the additional presence of phosphate, perhaps due to phosphate
binding at the UreG P-loop motif. Note, however, that when purified UreG was .
subjected to gel filtration chromatogaphy in the presence of 20% glycerol and 20 mM
phosphate, UreG was still present as a monomer. Several different types of columns were
examined for their abilities to purify UreG. Despite possessing a P-loop motif, UreG did
not bind to ATP- or GTP-linked agarose resins. Similarly, an Affi-Gel blue gel (BioRad)

column which has been found to bind several nucleotide-requiring proteins did not bind

113
the protein. Although DEAE-Sepharose and Mono Q ion exchange resins were

successfully used for UreG isolation, the protein tended to smear when chromatogaphed
on anionic exchangers.

Further characterization of purified UreG focused on its possible involvement in
providing CO2 for carbamylation of Lys-217, which then serves as a nickel ligand.
Specifically, I tested UreG for the presence of biotin and carbonic anhydrase activity.
Neither biotin, a carrier of C02 (reviewed in 27), nor carbonic anhydrase activity was
associated with purified UreG from E. coli cells containing pKAUG-l. One caveat to
note is that the UreG tested in these experiments was produced independently of the other
urease structural and accessory proteins. Possibly one or more of the additional accessory
proteins, such as UreF, must be co-produced in gowing cells in order to supply or link
biotin to UreG or to result in generation of carbonic anhydrase activity associated with

this protein.

The requirement of the P-loop motif for UreG function. Although UreG contains a P-
loop motif, which is commonly found in nucleotide-binding proteins (8), I was unable to
detect nucleotide binding to or hydrolysis by this protein. This result contrasts with the
situation found in the E. coli HypB protein (10), to which UreG exhibits 25 percent
identity. E. coli HypB binds and hydrolyzes GTP, likely related to its suggested role in
nickel ion processing (1 1). It should be noted that HypB from Rhizabium leguminasarum
does not bind or hydrolyze GTP or ATP (28), even though its proposed nucleotide

binding domain is absolutely conserved. In the cases of the R. leguminasarum HypB and

114

the K. aerogenes UreG, it is possible that additional accessory proteins must be present
for nucleotide binding and hydrolysis to be observed. An appealing model is that the
UreG P-loop motif is involved in the observed in viva energy requirement for urease
activation.

To further assess the importance of UreG P-loop motif, site-directed mutagenesis
was performed to replace either of two conserved residues (Lys-20 and Thr-21), acting to
bind the Mg-nucleotide complex, by Ala. E. coli cells containing the urease gene cluster
with these mutations and gown in the presence of nickel lacked urease activity. The cell
extracts were activated in vitro, presmnably from D-Apo or DF-Apo complexes which are
unaffected in the UreG P-loop variants. Using immunoblot analysis, 1 demonstrated that
DFG-Apo complexes were not present at detectable levels in soluble extracts of the P-
loop variants. In summary, these data indicate that a functional P-loop motif in UreG is
essential for in viva incorporation of nickel into the urease active site and may be

important for DFG-Apo formation or stability.

The DFG complex. A complex containing 1 UreD and UreF, per UreG protein was
detected in E. coli cells carrying the urease gene cluster with deletions in the urease
subunits and ureE genes. This complex was observed in the insoluble fraction upon cell
lysis and centrifugation, but was solubilized upon addition of detergent to this protein
pool. Following DEAE-Sepharose and Extracti-Gel D chromatogaphies, DF G was
shown to bind to an ATP-linked agarose resin. A P-loop variant in UreG did not affect

formation of the DFG complex, indicating that nucleotide binding is not required for

115
UreD, UreF, and UreG association. The complex did not, however, bind to the ATP-

linked agarose resin. This data supports the idea that a functional UreG P-loop is
essential for nucleotide-binding of the DFG complex, but is not essential for its
formation.

Although the DF G complex was observed under highly artificial conditions and I
have not demonstrated the presence of such a species in cells containing the wild-type
urease gene cluster, these studies may provide sigrificant clues related to the in viva
urease metallocenter assembly mechanism. For example, DFG-Apo, the urease
apoprotein complex that has been proposed to be the key species needed for in viva
activation (5), could be formed by the association of urease apoprotein with the DFG
complex, rather than by a stepwise assembly of Apo to D-Apo (3), to DF-Apo (4), to
DFG-Apo (5) species. The evidence that DFG complex binds nucleotides via its P-loop
provides strong support that nucleotide-dependent metallocenter assembly will be

associated with the DFG-Apo species.

 

10.

1 16
REFERENCES

Lee, M. H., Mulrooney, S. B., Renner, M. J., Markowicz, Y. & Hausinger, R.
P. (1992). Sequence of ureD and demonstration that four accessory genes (ureD,

ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis. Journal
of Bacteriology 174, 4324-4330.

Lee, M. H., Pankratz, H. S., Wang, S., Scott, R. A., Finnegan, M. G.,
Johnson, M. K., Ippolito, J. A., Christianson, D. W. & Hausinger, R. P.
(1993). Purification and characterization of Klebsiella aerogenes UreE protein: a
nickel-binding protein that functions in urease metallocenter assembly. Protein
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Park, I.-S., Carr, M. B. & Hausinger, R. P. (1994). In vitro activation of urease
apoprotein and role of UreD as a chaperone required for nickel metallocenter
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Moncrief, M. B. C. & Hausinger, R. P. (1996). Purification and activation
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Park, I.-S. & Hausinger, R. P. (1995). Evidence for the presence of urease
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Park, I.-S. & Hausinger, R. P. (1995). Requirement of carbon dioxide for in
vitro assembly of the urease nickel metallocenter. Science 267, 1156-1158.

Jabri, E., Carr, M. B., Hausinger, R. P. & Karplus, P. A. (1995). The crystal
structure of urease from Klebsiella aerogenes. Science 268, 998-1004.

Saraste, M., Sibbald, P. R. & Wittinghofer, A. (1990). The P-loop-a common
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Lee, M. H., Mulrooney, S. B. & Hausinger, R. P. (1990). Purification,
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Lutz, A., Jacobi, A., Schlensog, V., Btihm, R., Sawers, G. & Btick, A. (1991).
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Park, I.-S. (1994). Thesis. East Lansing, Nfl: Michigan State University.

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CHAPTER 6

PURIFICATION OF UREF AND CHARACTERIZATION OF NON-NATIVE

URED-UREF-UREG—UREASE APOPROTEIN COMPLEXES

119

120
ABSTRACT

Urease nickel metallocenter assembly in Klebsiella aerogenes requires the presence of
UreD, UreF, and UreG accessory proteins and is further facilitated by UreE. Urease
apoprotein exists in an uncomplexed form as well as in a series of complexes including a
UreD-UreF-UreG-urease apoprotein complex (I.-S Park, and R. P. Hausinger, J. Bacteriol.
177:1947-1951, 1995). In this study, ureF was overexpressed in the absence and presence
of the other urease genes. UreF was shown to be highly insoluble in the absence of the
other urease components and required detergent for solubilization. In contrast, when
produced as part of a thioredoxin-UreF fusion protein, the sample was soluble and was
readily purified. Overexpression of ureF along with the other urease genes resulted in
formation of a UreD-UreF-UreG-urease apoprotein species that was distinct in
chromatogaphic and electrophoretic behavior from the in viva species identified earlier.
The final level of activation, the nickel ion and bicarbonate dependencies, and the metal ion
inactivation properties of this species were not enhanced over the properties observed for
urease and UreD-urease apoprotein complexes. I conclude that the UreD-UreF-UreG-
urease apoprotein studied in this paper is unlike the form involved in urease activation in

viva.

121
INTRODUCTION

Urease nickel metallocenter assembly in Klebsiella aerogenes is a complicated
process that requires three accessory proteins (UreD, UreF, and UreG) and is facilitated
by a fourth protein (UreE) (reviewed in 1). In addition to the accessory protein
requirement, CO2 is necessary to bridge the two active site nickels in the form of a
carbamylated lysine (2). The accessory proteins are encoded in the same gene cluster as
the three urease structural genes (ureA, ureB, ureC). The roles of the accessory proteins
in metallocenter assembly are unclear. UreE is a nickel-binding protein (3) and is thought
to function as a nickel donor. The three required accessory proteins participate in several
complexes that contain UreD and urease apoprotein (D-Apo) (4), UreD, UreF, and urease
apoprotein (DF-Apo) (5, Chapter 4), UreD, UreF, UreG, and urease apoprotein (DFG-
Apo) (6), or a complex containing UreD, UreF, and UreG (DFG) (Chapter 5). UreF,
when present in the DF-Apo complex, precludes nickel from binding to the urease active
site until Lys-217 is carbamylated. UreG contains a P-loop motif, typically found in
nucleotide-binding proteins, and suggestive evidence indicates that this motif is essential
for in viva incorporation of nickel into the active site (Chapter 5).

In order to better understand the in viva metallocenter assembly process for urease, I
enhanced cellular synthesis of UreF in the absence and the presence of the other mease
components. These studies were desigred to facilitate the purification and characterization
of UreF and DFG-Apo. I have found that UreF is highly insoluble in the absence of the
other urease components, and I’ve overcome this limitation by causing the cells to

synthesize a thioredoxin-UreF fusion protein that I have purified. Ftnthermore, I have

 

122

shown that overexpression of ureF along with the other urease genes led to the formation of
DFG-Apo complexes that are distinct in its properties from those described earlier (6) and is

probably not relevant to the cellular activation process.

MATERIALS AND METHODS

Bacterial strains and plasmid construction. All molecular biology methods followed
the general methods outlined in Sambrook et a1. (8). Plasmid pKAUD2F+, containing
overexpressed ureF and a mutated ureD promoter region to allow overexpression of this
gene, has been described previously (5). This plasmid was transformed into Escherichia
coli DHSor.

To facilitate UreF purification, three methods were examined to optimize ureF
expression. The 1.68-kbp SalI fragnent of pKAU17 (9) was blunt ended with Klenow
fiagnent and ligated to phosphorylated Ndel linker (5'-ACCATATG-3'). The resulting
product was digested with Ndel and Aer and the 670-bp fragnent was ligated to the 5.7-
kbp Ndel-Nhel fiagnent of pET-l la (Novagen, Madison, WI), thus placing ureF under the
control of a T7 promoter. This construct, pETF, was transformed into E. coli BL21(DE3)
(N ovagen). Alternatively, a ureF gene fusion with a 5' terminal extension was generated by
blunt-ending the 670-bp Sall-Blnl fiagnent of pKAU17 with Klenow fiagnent and
inserting it into the 3.4-kbp BamHI-digested and blunt-ended vector pQE-32 (Qiagen,
Chatsworth, CA). This construct, pQEF, was transformed into E. coli M15[pREP4].

Finally, the 670-bp SalI-AvrII fragnent of pKAUl 7 was ligated to the 4.4-kbp XhaI-Xbal

 

123
fragnent of pThioHis C (Invitrogen, San Diego, CA). This construct, pThioHisF, was

transformed into E. coli DHSa.

Culture conditions and cell disruption. All cultures were gown in Ltuia-Bertani
(LB) broth supplemented with 100 pg ampicillin per ml. E. coli cells containing pETF or
pThioHisF were gown at 37 °C until they reached an OD6oo of ~0.6. Isopropyl B-D-
thiogalactopyranoside (IPT G) was added (1 mM final concentration) and the cells were
gown for an additional 3-5 h at 30 or 37 °C. The cells were harvested by centrifugation
and suspended in HEDG (25 mM HEPES [pH 7.4], 1 mM EDTA, 1 mM dithiothreitol, and
20% glycerol) buffer for cells containing pETF or in binding (20 mM sodium phosphate
[pH 7.8], 500 mM NaCl, 10% glycerol) buffer for cells with pThioHisF. E. coli
M15[pREP4] cells containing pQEF were gown in LB at 30 °C until an OD600 of «0.7-0.9,
induced with 2 mM IPT G, and cultured for an additional 5 hours. Cells were harvested by
centrifugation and suspended in PNC (20 mM NaI-IPO4 [pH 8.0], and 300 mM NaCl)
buffer. E. coli DH5a(pKAUD2F+) was gown at 30 or 37 °C to late stationary phase,
harvested by centrifugation, and suspended in PEDG (18 mM potassium phosphate [pH
7.4], 0.09 mM EDTA, 0.09 mM dithiothreitol, and 10% glycerol) buffer. Resuspended
cells were disrupted by 2-3 passages through a French pressure cell at 18,000 lb/in2 (l lb/in2
= 6.89 kPa), supplemented with 1 mM phenylmethylsulfonyl fluoride, and separated into

extracts and pellet fi'actions by centrifirgation at 100,000 x g for 45 min at 4 °C.

 

124
Partial purification of UreF. UreF was enriched fiom disrupted cell pellets of a

150 ml culture of E. coli BL21(DE3) containing pETF. The pellet was washed with 20 ml
of HEDG buffer, centrifuged at 26,000 x g for 45 minutes, and the supernatant was
removed. The sample was suspended in 20 ml HEDG buffer containing 0.5% (w/v) Triton
X-100 and incubated at 37 °C for 30 min or at 4 °C for 17 h. After centrifugation at 26,000
x g for 20 min, the supernatant fiaction containing UreF was added to a 001mm of Extracti-
Gel D Detergent Removing Gel (1.7 x 7 cm; Pierce, Rockford, IL). The flow-through
fractions containing UreF protein were concentrated and subjected to Superose 12
chromatogaphy (1.6 x 48.5 cm; Pharmacia Biotech, Piscataway, NJ) in HEDG buffer
containing 0.1 M KCl.

His-tagged, UreF fusion protein was enriched fiom disrupted cell pellets of a 100 ml
culture of E. coli M15[pREP4] carrying pQEF. The cell pellet was resuspended in PNC
buffer containing 0.5% Triton X-100 and solubilized as described above. The supematant
fraction was applied to a nickel-charged nitrilotriacetic acid resin (1.7 x 2 cm; Qiagen).
After washing the column with 500 ml of PNC buffer supplemented with 0.5% Triton X-
100 and 10% glycerol, the bound proteins were eluted from the column with PNC buffer
containing 1 M imidazole.

UreF fused to a thioredoxin protein was purified fi'om a 2-liter culture of E. coli
carrying pThioHisF. Cell extracts were chromatogaphed on a ProBond nickel-charged
Sepharose resin (1.5 x 6.8 cm, Invitrogen) equilibrated in binding bufier. The coltunn was
washed with binding bufier until the A230 was less than 0.01. The column was then washed

with 20 mM sodium phosphate (pH 6.0), 500 mM NaCl, and 10% glycerol until the A230

125
was less than 0.01. The column was washed with 50 mls of binding buffer to raise the pH

back up to pH 7.8, and then proteins were eluted with binding buffer containing 50 mM
imidazole. Fractions containing ThioHisF were pooled and dialyzed against 20 mM sodium
phosphate (pH 7.8), 0.1 mM dithiothreitol, and 10% glycerol. The sample was applied to a
Mono Q HR 10/ 10 (Pharmacia Biotech) column equilibrated with the same buffer. The
proteins were eluted with a linear salt gadient to 1 M KCl in the same buffer. Fractions
containing ThioHisF were pooled and reapplied to the ProBond resin and
chromatogaphed again as described above to remove proteins that bound nonspecifically

to the initial ProBond column.

Enrichment of DFG-Apo. Cell extracts fiom a 3-liter culture of E. coli
DH5a(pKAUD2F+) were used to enrich a series of DFG-Apo complexes. The sample was
applied to a DEAE-Sepharose column (2.5 x 19 cm; Pharmacia Biotech) equilibrated in
PEDG buffer, and proteins were eluted from the colmnn by using a 400-ml linear salt
gadient to 0.5 M KCl in the same buffer. Fractions containing the DFG-Apo were pooled,
dialyzed against PEDG buffer, and applied to a Mono Q HR 10/ 10 (Pharmacia Biotech)
column equilibrated with the same buffer. The proteins were eluted with a linear salt
gadient to l M KCl in PEDG buffer. DFG-Apo eluted hour the column at 04-06 M
KCl. Fractions containing the desired proteins were pooled, dialyzed against PEDG
containing 0.1 M KCl. The protein pool was concentrated in a Centriprep-lO or -30
(Amieon, Beverly, MA) to 4 mls and chromatogaphed, via 2 runs, on a Superose 6

column (Pharmacia Biotech, 1.6 x 49 cm) equilibrated in the same buffer.

 

Polyacrylamide gel electrophoresis. Sodium dodecyl sulfate (SDS)-
polyacrylamide gel electrophoresis was carried out by using the buffers described by
Laemmli (10) and included 4.5% and 12.5% polyacrylamide stacking and running gels.
Nondenaturing gels used the same buffers without detergent and consisted of 3% and 6%
polyacrylamide stacking and running gels. The gels were either stained with Coomassie
brilliant blue or electroblotted onto nitrocellulose, probed with anti-K aerogenes urease
antibodies (9), and visualized by using anti-rabbit immunoglobulin G-alkaline phosphatase
conjugates (11). When testing for the presence of biotin, blots were probed and detected
with ExtrAvidin-alkaline phosphatase (Signa Chemical). The band intensities of
Coomassie-stained gels were determined with an AMBIS (San Diego, CA) gel scanner. For
calculation of the ratios of UreD, UreF, UreG, and UreC, M, values of 29,300, 27,000,

21,800 and 60,300 were used.

Urease activity assays. Urease activity was measured by quantitating the rate of
armnonia release from urea by formation of indophenol, which was monitored at 625 nm
(12). One unit of urease activity was defined as the amount of enzyme required to
hydrolyze l pmole of urea per min at 37 °C. The standard assay buffer consisted of 25 mM
HEPES (pH 7.75), 0.5 mM EDTA, and 50 mM urea. Protein concentrations were
determined by using a commercial assay (Bio-Rad, Hercules, CA) with a bovine serum

albumin standard.

 

127

Activation of urease apoprotein. Routine urease apoprotein activation buffer
consisted of 100 mM HEPES (pH 8.3), 150 mM NaCl, 100 mM NaHCO;, and 100 pM
NiCl2 (2). For specific experiments, the conditions were modified by varying the
bicarbonate or nickel ion concentrations, adding other metal ions to the activation buffer, or

incubating samples in the presence of metal ions prior to addition of bicarbonate.

RESULTS AND DISCUSSION

High level synthesis and partial purification of UreF. UreF, a protein that is
absolutely required for in viva activation of urease (reviewed in 1), has not been purified or
characterized from any microorganism. K aerogenes UreF purification has been
confounded by the low expression level of the corresponding gene in the wild type
microorganism and in E. coli cells carrying the K. aerogenes urease gene cluster (13).
Analogous to the situation for ureD, another essential urease accessory protein, ureF,
possesses a non-consensus ribosome binding site. Unlike the case with ureD (4), however,
alterations to the ureF ribosome binding site by site-directed mutagenesis did not result in
the synthesis of high levels of the gene product (14). Three alternative strategies were
devised to allow high level synthesis of the ureF gene product in the absence of the other
urease components.

In E. coli(pETF), ureF expression was regulated through an IPTG-inducible T7
RNA polymerase acting on a T7 promoter located immediately upstream from the cloned

gene. When induced with IPTG, high level synthesis of UreF was observed (Fig. 1, lane 2).

128

The desired protein, however, was not in the soluble cell extracts (Fig. 1, lane 3). In the
case of cells gown at 30 °C, but not for 37 °C-gown cells, about 50 % of UreF was
solubilized fiom the pellet by incubation in buffer containing 0.5% Triton X-100 (Fig. 1,
lane 4). The identity of the predominant band was confirmed to be UreF by N-terminal
amino acid sequence analysis (data not shown). Upon removal of detergent, the solubility
of UreF decreased and firrther purification efforts were hindered. The protein sample
appeared to smear during chromatogaphy on ion exchange or hydrophobic columns. Size
exclusion chromatogaphy revealed the presence of an aggegated form containing UreF as
well as other peptides.

An alternative approach to UreF purification made use of E. coli M15[pREP4] cells
canying pQEF where ureF expression is controlled by an IPTG-regulated T5 promoter. In
this construct, UreF was synthesized at high levels as a fusion protein with an N-terminal
extension containing six histidine residues. The presence of the His-tag did not enhance
solubility of UreF and the fusion protein was found in the pellet fiaction after cell lysis and
centrifugation (data not shown). As observed for the pETF-encoded UreF protein, Triton
X- 1 00 treatment solubilized a portion of His-tagged UreF. The His-tag was ineffective for
protein purification when using a nickel-charged nitrilotriacetic acid column, possibly due
to aggegation of proteins in the sample and the propensity of UreF to precipitate out of
solution.

A third construct containing ureF was generated by creating a gene fusion of a
thioredoxin gene containing a nickel binding site and ureF. This ThioHisF fusion protein

was observed in cell extracts and was purified using a nickel-amnity and anion exchange

 

M (kD)
97.4-
66.2-

45-

-UreF

14.4-

 

Figure l. SDS/polyacrylamide gel electrophoretic analysis of UreF solubilization. Samples
included: lane 1, molecular weight markers (phosphorylase b, M, 97,400; bovine serum
albumin, M, 66,200; ovalbumin, M, 45,000; carbonic anhydrase, M, 31,000; soybean
trypsin inhibitor, M, 21,000; and lysozyme, M, 14,400); lane 2, disrupted cell suspension
from E. coli BL21(DE3)[pETF]; lane 3, soluble cell extracts; lane 4, solubilized sample
after treating lysed cell pellets with HEDG buffer containing 0.5% Triton X-100; lane 5,

pellet remaining after partial solubilization of UreF.

130

1 2 3
974-”

66.2- " ”W

w -ThioHisF

Figure 2. Purification of ThioHisF as analyzed by SDS/polyacrylamide gel electrophoresis.
Samples included: lane 1, molecular weight markers; lane 2, first ProBond ThioHisF pool;

lane 3, second ProBond ThioHisF pool.

131
chromatogaphies. Chromatogaphy on the nickel-affinity column resulted in partial

purification of ThioHisF (Fig. 2, lane 2). The bound proteins eluted from the column at a
very low imidazole concentration (< 50 mM) so purification of ThioHisF using an
imidazole gadient was not very helpful. ThioHisF was chromatogaphed on a Mono Q
column to remove the protein, labelled X (Fig. 2, lane 2), that co-eluted with ThioHisF off
the ProBond column. Mono Q fractions containing ThioHisF were pooled and reapplied to
the nickel-affinity resin and purified as described for the first ProBond chromatogaphy
step. This was done to remove proteins that bound non-specifically during the first
ProBond chromatogaphy procedure. The ThioHisF protein was reasonably pure (Fig. 2,
lane 3) and remained soluble throughout the purification procedure. Since high levels of
CO2 are known to be required for carbamylation of Lys-217 in the urease apoprotein (2)
in order to form the binuclear active site (15), I tested whether the UreF fusion protein
possessed a cofactor that could function to deliver C02. The ThioHisF protein did not
contain biotin when analyzed by Western blot (immunoblot) analysis using avidin-linked
alkaline phosphatase (data not shown). The fusion protein does, however, contain an
enterokinase cleavage site that was used to release UreF from the thioredoxin. Protease
treatment resulted in cleavage of thioredoxin from UreF, and UreF remained soluble. After
gel filtration to remove the enterokinase protein (M, 43,000) and the thioredoxin fusion
partner (M, 14,400), UreF (M, 27,000) may then be used to make antibodies or be used for

crystallization trials. This method provides the first soluble purification scheme for UreF.

 

132

Enrichment of DFG-Apo. A series of urease apoprotein complexes containing
UreD, UreF, and UreG were previously described (6), but these species were present at
levels too low to allow purification and detailed characterization. Because the prior
complexes were observed in cells that synthesized high levels of urease subunits, UreD, and
UreG, the limiting component for their formation appeared to be UreF. In an efi‘ort to
optimize synthesis of these complexes, I constructed plasmid pKAUD2F+ and used it to
purify DFG-Apo.

E. coli(pKAUD2F+) cells synthesized high levels of urease, UreD, UreF, and UreG.
The four proteins were soluble in extracts and co-eluted during purification using DEAE-
Sepharose, Mono Q, and Superose 6 chromatogaphies (Fig. 3). Size exclusion
chromatogaphy results, however, indicated the presence of multiple, very large species (M,
> 670,000) that eluted at and near the void volume of the Superose 6 column, in contrast to
the reported behavior for DFG-Apo (6). Nondenaturing gel electrophoretic analysis
revealed that much of the protein did not enter the running gel and the protein that did
electrophorese Irrigated very slowly (data not shown), again differing from the DFG-Apo
properties (6). Due to these differences the species purified here are desigrated DFG-Apo*
to distinguish them from the DFG-Apo complexes observed in viva in cells not
overexpressing ureF. Based on gel scanning analysis of Coomassie blue-stained bands on
an SDS/polyacrylamide gel, there appeared to be an average of 1.16-1.19 UreD, 0.93-0.96
UreG, and 0.77-0.81 UreF per UreC protein in the DFG-Apo“ pool. Efforts to disrupt the
aggegated complex or to further resolve the complex fi'om contaminating proteins led to

diminishment in intensity

 

133

97.4- a
66-2- CH.- “UreC

14.4- “I

 

>UreA/UreB

Figure 3. Analysis of DFG-Apo complex purified from E. coli(pKAUD2F+) by

SDS/polyacrylamide gel electrophoresis. lane 1, molecular weight

Samples included:

markers; lane 2, Superose 6 pool.

134
of the UreG peptide compared to the UreD, UreG, and urease peptides when examined by

SDS/polyacrylamide gel analysis (data not shown).

The DFG-Apo* complexes were shown to be activation competent, yielding 450-
600 U/mg of total protein when using standard activation conditions that had been
optimized for activating D-Apo and Apo. The level and rate of activation of DFG-Apo*
complexes are similar to that observed for D-Apo (4). The bicarbonate (Fig. 4) and nickel
concentration (Fig. 5) dependencies of the DFG-Apo* were similar to those observed for
both Apo and D-Apo (2, 4), exhibiting half-maximal activation at ~25 mM added
bicarbonate and ~75 pM NiCl2. The ability of various metal ions to inhibit activation of
DFG-Apo was studied since previous work on Apo and D-Apo had demonstrated inhibition
by nickel ion as well as other divalent metals (16). Prior incubation of the DFG-Apo“ pool
with either 100 or 500 pM NiCl2 for 80 minutes resulted in a 40-70% or 50-80% decrease
respectively in the extent of urease activation when subsequently incubated in the presence
of both nickel ions and bicarbonate (data not shown). The extent of activation varied from
one purification to another, perhaps due to different levels of UreG in the complex pools.
Although the DFG-Apo* complexes are able to be activated in vitro, the final level of
activation and the nickel ion and bicarbonate dependencies for these complexes are not
enhanced over the properties observed for activation of Apo and D-Apo. I believe that the
present DFG-Apo* species are not sigrificant to urease metallocenter assembly. Rather, I
suspect that these species likely represent non-native aggegates with limited function for

urease assembly.

”L

135

 

 

 

500 —
7“

400 —
A e
on
Si 300 r =/
.3:
.2.
8 I '
< zoo -
O ’A‘
55
Q
d)
a.
CI)

100 —

0 ' I I i 1
0 100 150 200 250
Time (min)

Figure 4. Bicarbonate ion concentration dependence of activation for the DFG-Apo*. The
sample (0.5 mg/ml) was incubated at 37 °C in 100 mM HEPES (pH 8.3) buffer containing
150 mM NaCl, 100 pM NiCl2, and the following concentrations of NaHC03: 0 (O), 5 (I),

10 (A), 25 (7), 50(0), 75 (D), or 100 (A) mM added NaHCO3.

 

136

 

 

 

600 -
O
500 - I
O I
’5?) ._
g 400
3 #A
$3
.2 ‘7
ii
0
.‘E
O
o
o.
m
l O r O l

 

 

 

80 100 120 140

Time (min)

Figure 5. Nickel ion concentration dependence of activation for the DFG-Apo*. The
purified sample (0.1 mg/ml) was incubated at 37 °C in 100 mM HEPES (pH 8.3) buffer
containing 150 mM NaCl, 100 mM NaHCO3 and 0 (O), 25 (I), 50 (A), 75 (V), 100(0),

200 (El), 500 (A), or 1000 (V) pM NiCl2. Aliquots were removed at the indicated times

and the samples were assayed for urease activity.

 

 

10.

137
REFERENCES

Moncrief, M. B. C. & Hausinger, R. P. (1996). Nickel incorporation into
urease. In Mechanisms of Metallocenter Assembly, pp. 151-171. Edited by R. P.
Hausinger, G. L. Eichhom & L. G. Marzilli. New York: VCH Publishers.

Park, I.-S. & Hausinger, R. P. (1995). Requirement of carbon dioxide for in
vitro assembly of the urease nickel metallocenter. Science 267, 1156-1158.

Lee, M. H., Pankratz, H. S., Wang, S., Scott, R. A., Finnegan, M. G.,
Johnson, M. K., Ippolito, J. A., Christianson, D. W. & Hausinger, R. P.
(1993). Purification and characterization of Klebsiella aerogenes UreE protein: a
nickel-binding protein that functions in urease metallocenter assembly. Protein
Science 2, 1042-1052.

Park, I.-S., Carr, M. B. & Hausinger, R. P. (1994). In vitro activation of urease
apoprotein and role of UreD as a chaperone required for nickel metallocenter
assembly. Proceedings of the National Academy of Sciences 91, 3233-3237.

Moncrief, M. B. C. & Hausinger, R. P. (1996). Purification and activation
properties of UreD-UreF-urease apoprotein complexes. Journal afBacterialagy
178, 5417-5421.

Park, I.-S. & Hausinger, R. P. (1995). Evidence for the presence of urease
apoprotein complexes containing UreD, UreF, and UreG in cells that are
competent for in vivo enzyme activation. Journal of Bacteriology 177, 1947-
1951.

Lee, M. H., Mulrooney, S. B. & Hausinger, R. P. (1990). Purification,
characterization, and in vivo reconstitution of Klebsiella aerogenes urease
apoenzyme. Journal of Bacteriology 172, 4427-4431.

Sambrook, J., F ritsch, E. F. & Maniatis, T. (1989). Molecular cloning: a
laboratory manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory.

Mulrooney, S. B., Pankratz, H. S. & Hausinger, R. P. (1989). Regulation of
gene expression and cellular localization of cloned Klebsiella aerogenes (K.
pneumaniae) urease. Journal of General Microbiology 135, 1769-1776.

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature (London) 227 , 680-685.

ll.

12.

l3.

14.

15.

16.

138

Blake, M. S., Johnston, K. H., Russel-Jones, G. L. & Gotschlich, E. C. (1984).
A rapid, sensitive method for detection of alkaline phosphatase-conjugated
anti-antibody on western blots. Analytical Biochemistry 136, 175-179.

Weatherburn, M. W. ( 1967). Phenol-hypochlorite reaction for determination of
ammonia. Analytical Chemistry 39, 971-974.

Lee, M. H., Mulrooney, S. B., Renner, M. J., Markowicz, Y. & Hausinger, R.
P. (1992). Sequence of ureD and demonstration that four accessory genes (ureD,

ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis. Journal
of Bacteriology 174, 4324-4330.

Park, I.-S. (1994). Thesis. East Lansing, MI: Michigan State University.

Jabri, E., Carr, M. B., Hausinger, R. P. & Karplus, P. A. (1995). The crystal
structure of urease from Klebsiella aerogenes. Science 268, 998-1004.

Park, I.-S. & Hausinger, R. P. (1996). Metal ion interactions with urease and
UreD-urease apoproteins. Biochemistry 35, 5345-5352.

CHAPTER 7

ADDITIONAL STUDIES

139

140

A. Identification of a series of urease apoprotein forms. During routine purification of
urease apoprotein (termed Apo) from Escherichia coli pKAU22AureD (1), a unique
series of bands were observed when purified Apo was analyzed on a nondenaturing
polyacrylamide gel electrophoresis (Fig. 1, lane 2). When activated by the addition of
nickel and bicarbonate, the Apo species did not collapse completely. The four major
species remained while some of the larger less prevalent species disappeared (data not
shown). To determine how these species were formed, I tested for collapse of the Apo
forms by incubating purified protein for 1 hour at 37 °C in 18 mM phosphate (pH 7.3), 1
mM EDTA, 1 mM dithiothreitol (DTT) and various compounds such as KCl,
hydroxylamine, glycerol, and additional quantities DTT and EDTA. The incubated samples
were subjected to nondenaturing polyacrylamide gel electrophoresis (Fig. 1). DTT
collapsed the species down to the form typically observed for holo- or apoprotein (Fig. 1,
lane 7). One mM DTT was present at all stages of the Apo purification procedure, so it is
unclear why additional incubation with 10 mM DTT would collapse the species. Incubation
with glycerol (Fig. 1, lane 4), sodium bicarbonate (Fig. 1, lane 5), KCl (Fig. 1, lane 6),
EDTA (Fig. 1, lane 8), and hydroxylamine (data not shown) did not affect Apo species
formation. As observed with DT'I‘, 2-mercaptoethanol was also able to collapse the Apo
species. These results suggest that disulfide bonds may be involved in the Apo species
formation.

Since the data suggested that DTT might be reducing disulfide bonds in the Apo
species, I speculated that a particular Cys in the Apo might be involved. I decided to test for

Apo species formation using a plasmid containing a mutated Cys (C319A) at the active site

 

141

 

Figure l. Nondenaturing polyacrylamide gel electrophoretic analysis of Apo. Purified Apo
was incubated in 18 mM phosphate (pH 7.4), 0.1 M KCl, 0.09 mM EDTA, 1 mM
dithiothreitol with various compounds at 37 °C for 1 hour then analyzed on a 6% native
polyacrylamide gel. Samples included: lane 1, D-Apo complexes; lane 2, Apo not
incubated at 37 °C; lane 3, Apo only; lane 4, Apo with 20% glycerol; lane 5, Apo with 150
mM NaHCO;; lane 6, Apo with 0.4 M KCl; lane 7, Apo with 1 mM dithiothreitol; lane 8,

Apo with 10 mM EDTA.

 

142
(3). The plasmid containing C319A was chosen because Cys-319 is the only Cys located in

the active site and it was mutagenized previously. The presence of a flexible loop over the
active site (4) would support the hypothesis that Cys 319 could be interacting with another
Cys located outside of the active site. I removed ureD from the plasmid to prevent possible
D-Apo complexes from forming. The plasmid containing a pKAUl7C319A ureD deletion
mutant was constructed by religating the 7.5-kbp NruI-Sacl blunt-ended fiagnent of
pKAUl7C319A and transforming E. coli DHSa. When the C319 purified Apo was
analyzed by nondenaturing polyacrylamide gel electrophoresis, the species pattern observed
previously for wild-type Apo was not detected (Fig. 2, lane 2). Instead, bands were detected
that corresponded to the expected Apo/holoprotein band position (labelled Apo), but also
additional bands (labelled X*) were detected. The bands labelled X* electrophoresed at the
same position where bands were observed previously when DF-Apo complexes were
activated with nickel and bicarbonate. These bands were labelled X (5, Chapter 6, Fig. 3C)
and were found to contain urease when analyzed by 2-dimensional polyacrylamide gel
electrophoresis. The bands observed with C319A Apo were also found to contain urease
(data not shown), and incubation with 10-100 mM DTT did not alter the C319A Apo
species. This suggests that Cys-319 may play a role in these species formation possibly by

disulfide formation and these results might explain why dithiothreitol collapses the species.

It was puzzling why these species are forming and why the crystal structure of Apo
didn’t identify any difference between holourease and Apo with the exception of nickel
being absent from the active site and Lys-217 not being carbamylated (6). Apo utilized to

make the crystals was purified from cells containing all the accessory proteins. When Apo

143
was purified from E. coli(pKAU22), these Apo species were not detected and a single Apo

band was detected on native gels (data not shown). When the additional Apo species were
observed, Apo was purified from a ureD gene deletion mutant (Fig. l). UreD could still
possibly have a role as a chaperonin involved in preventing these species from forming.
UreD may prevent disulfide bond formations with Cys-319 by stabilizing or controlling the
flexible loop’s structure over the active site. One could imagine UreD functioning as a
doorman and controlling the influx of essential molecules such as nickel and bicarbonate
while preventing unwanted guests such as other metal ions and potentially reactive
sulfltydryl goups. Evidence for this is supported from studies showing that the presence of
UreD in the UreD-Apo complexes appears to reduce the rate of Apo interaction with nickel
as well as other metal ions (7). Due to the absence of UreD in the cell, these Apo species
may be inactive forms of urease that may contain disulfide goups and may also contain
nonproductive-bound nickel or other metal ions. Cross-linking studies have shown that
UreD interacts with the [3 subunit of urease. The active site is located in the a subunit, but
when looking at the crystal structure, [(0437);], UreD associated with the [3 subunit of one
((1137) trirner may interact with the or subunit of the adjacent trirner. Crystallization of the
D-Apo complexes will undoubtedly provide insight into UreD’s role in metallocenter

assembly.

 

Figure 2. Nondenaturing polyacrylamide gel electrophoretic analysis of purified Apo
from E. coli(pKAUl7C319AAureD). Samples include; lane 1, D-Apo complexes, and

lane 2, purified C319A Apo.

145
B. Incubation of Apo and UreD-UreF-UreG complex. In Chapter 6, I attempted to

purify the UreD-UreF-UreG-Apo (termed DFG-Apo) species by overexpressing ureF in a
strain already overexpressing ureD. Unfortunately, the complexes observed (termed
DFG-Apo*) were not the species observed in viva (2) and are probably not relevant to the
cellular activation process. I wanted to test the theory that UreD-UreF-UreG (DFG)
complex might bind to Apo to form the in viva DFG-Apo. The urease metallocenter
assembly model suggests that the DFG-Apo species might be constructed in this manner.
I attempted to form the in viva DFG-Apo species by incubating purified Apo and partially
purified DF G together. The sample was incubated overnight at 4 °C and then subjected to
Superose 12 chromatogaphy. Fractions off the column were analyzed by denaturing
polyacrylamide gel electrophoresis (Fig. 3, lanes 3-7). DFG and some Apo co-eluted
during gel filtration. [Note: When ATP was included in the sample, there was no
detectable difference in the Superose 12 chromatogam or protein banding patterns on
native and denaturing gels (data not shown)]. Fractions were also analyzed by
nondenaturing polyacrylamide gel electrophoresis, transferred to nitrocellulose, and
analyzed by immunoblot analysis visualized with anti K. aerogenes urease antibodies
(Fig. 4). Samples containing Apo, UreD, UreF, and UreG contained a band (labelled X,
Fig. 4, lanes 4-5) that migated at a position more closely resembling the DFG-Apo
species observed in viva. This band was not detected when Apo and DFG samples were
analyzed separately by immunoblot analysis (data not shown). Interestingly, a band that
migated at approximately the same position as lD-Apo was also observed (Fig. 4, lanes

4-6). There are two possible explanations for formation of lD-Apo. First, DFG-Apo

146

 

Figure 3. SDS/polyacrylamide electrophoretic analysis of Apo and DFG pool after
Superose 12 chromatogaphy. Samples included: lane 1, molecular weight markers
(phosphorylase b, M, 97,400; bovine serum albumin, M, 66,200; ovalbumin, M, 45,000;
carbonic anhydrase, M, 31,000; soybean uypsin inhibitor, M, 21,000; and lysozyme, M,
14,400); lane 2, DFG-Apo“; lanes 3-7, successive Superose 12 fractions of the Apo and

DFG mixture.

147

 

Figure 4. Native polyacrylamide gel electrophoretic analysis of Apo and DFG pool after
Superose 12 chromatogaphy. Western blot analysis of a nondenaturing polyacrylamide
gel visualized with anti-Klebsiella aerogenes urease antibodies. Samples included: lane

1, D-Apo; lane 2, Apo; lanes 3-7, same samples analyzed in Fig. 2, lanes 3-7.

148

may have formed, but UreF and UreG dissociated from the complex resulting in D-Apo.
This seems unlikely because one would expect to see 3D- and 2D-Apo complexes also.
Another possibility is that some UreD dissociated fi'om DFG and bound to Apo to form
lD-Apo. I did not perform immunoblot analysis with anti-K. aerogenes UreD antibodies
to be sure this band contains UreD. Addition of DFG to Apo provides evidence
supporting the model that Apo and DF G can interact to form DFG-Apo, at least in vitro.
This procedure may be utilized in the future to characterize DFG-Apo since
overexpression of ureD, ureF, ureG, and the urease structural subunits (plasmid
pKAUD2F+) produces an anomalous DFG-Apo* species in viva (Chapter 6).

Since formation of the in viva DFG-Apo complexes in vitro appeared to be a
possible prospect with Apo and DFG complexes, 1 tested for formation of the DFG-Apo
complexes by incubating the DF-Apo complexes (5) with partially purified UreG under
the same experimental conditions used for the Apo and the DF G complexes. When
analyzed by gel filtration chromatogaphy, association of DF-Apo and UreG did occur
(data not shown), but the complexes observed were the multiple, very large species
observed with the DFG-Apo* complexes in E. coli(pKAUD2F+) (Chapter 6). These
results suggest that stepwise addition of accessory proteins to Apo in vitro may not form
the DFG-Apo observed in vivo, and formation of the DFG complex followed by
incubation with Apo may be the most logical approach in vitro to form the DFG-Apo

complexes.

149
REFERENCES

Lee, M. H., Mulrooney, S. B., Renner, M. J., Markowicz, Y. & Hausinger, R.
P. (1992). Sequence of ureD and demonstration that four accessory genes (ureD,

ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis. Journal
of Bacterialagz 174, 4324-4330.

Park, I.-S. & Hausinger, R. P. (1995). Evidence for the presence of urease
apoprotein complexes containing UreD, UreF, and UreG in cells that are
competent for in vivo enzyme activation. Journal of Bacteriology 177 , 1947-
1951.

Martin, P. R. & Hausinger, R. P. (1992). Site-directed mutagenesis of the active
site cysteine in Klebsiella aerogenes urease. Journal of Biological Chemistry 267 ,
20024-20027.

Jabri, E., Carr, M. B., Hausinger, R. P. & Karplus, P. A. (1995). The crystal
structure of urease from Klebsiella aerogenes. Science 268, 998-1004.

Moncrief, M. B. C. & Hausinger, R. P. (1996). Purification and activation
properties of UreD-UreF-urease apoprotein complexes. Journal of Bacteriology
178, 5417-5421.

Jabri, E. & Karplus, P. A. (1996). Structures of the Klebsiella aerogenes urease
apoenzyme and two active-site mutants. Biochemistry 35, 10616-10626.

Park, I.-S. & Hausinger, R. P. (1996). Metal ion interactions with urease and
UreD-urease apoproteins. Biochemistry 35, 5345-5352.

CHAPTER 8

CONCLUSIONS AND FUTURE PROSPECTS

150

151

The initial goal of this work was to try and characterize two of the accessory
proteins, UreG and UreF, required for urease metallocenter assembly in Klebsiella
aerogenes. Although I was unable to fully characterize both of these proteins
individually, I was able to identify several complexes in which these proteins play an
important role. Characterization of these complexes helped to identify the roles of these
proteins in the context of all the protein players in this complicated production.

Crystallization of urease was essential for resolving the structure of the
holoenzyme, and its active site environment. I provided urease and Seleno-methionine-
containing urease to Dr. Andy Karplus for crystallogaphic analysis. The X-ray crystal
structure analysis of K. aerogenes has been resolved to 2.2 A. The enzyme fimctions as a
tightly packed trirner of trimers [(orBy)3] containing three bi-nickel active sites. The two
nickel ions per active site are located 3.5 A apart and are bridged by a carbamate formed
on Lys-217. A solvent molecule is tightly bound to one metal ion and appears to partially
bridge to the second. One nickel ion possesses a distorted trigonal bipyramidyl or
distorted square pyramidyl geometry and the five ligands include His-134, His-136, Asp-
360, Lys-217 carbamate, and water (or hydroxide). The second nickel ion possesses
pseudotetrahedral geometry and is liganded by His-246, His-272, Lys-217 carbamate, and
solvent in partial occupancy. In addition to the metallocenter ligands, the crystal structure
reveals several additional residues worth noting. His-219 is appropriately positioned to
stabilize substrate binding to the fourth ligand position of the second nickel ion. His-320
is located near the bi-nickel center and is likely to participate in catalysis. Finally, Cys-

319 is within the active site environment, but does not play a catalytic role.

152

Catalysis has been suggested to proceed by urea binding in O-coordination to the
fourth coordination site of the second nickel ion with stabilization rendered by His-219.
The solvent molecule that is now solely coordinated to the first nickel ion is thought to
carry out nucleophilic attacks on the bound urea to form a tetrahedral intermediate.
Elimination of ammonia from the intermediate is facilitated by proton donation from His-
320 acting as a general acid, and carbamate is released.

My studies on the activity of urease in the crystalline state found that the enzyme
has less than 0.05% of the activity observed for the soluble enzyme under standard assay
conditions. The trace amounts of activity observed arise from limited enzyme activity at
the crystal surfaces or trace levels of enzyme dissolved into the crystal storage buffer.
This data support the necessity for a conformational change during urea catalysis.

l overexpressed ureF and partially purified UreF in its insoluble form using Triton
X-100. UreF was also purified as part of a thioredoxin fusion protein which was found in
the soluble portion of the cell, unlike wild-type UreF. Overexpression of ureF and ureD
in a ureG deletion mutant resulted in production of a series of UreD-UreF-urease
apoprotein (DF-Apo) complexes. These complexes exhibited activation properties
distinct fi'om urease (Apo) and UreD-urease (D-Apo) apoprotein complexes. These
complexes were resistant to inactivation by NiCl2 and exhibited a decreased bicarbonate
requirement. Irnmunoblot analysis with polyclonal anti-urease and anti-UreD antibodies
indicated that in these complexes UreD is masked, presumably by UreF, and the DF-Apo
complexes migate at the same positions as D-Apo complexes during nondenaturing

polyacrylamide gel electrophoresis. One of the most interesting results from the DF-Apo

153

characterization studies is the ability of the complexes to preclude nickel ions from the
active site but the inability to preclude other divalent metal ions. The mechanism by
which DF-Apo can distinguish between the nickel and other metal ions is unknown.
Once carbamylation of Lys-217 has occurred, UreF may detect this and conformational
change occurs within the complex and Ni ion is permitted to enter the active site to form
the urease metallocenter. The D-Apo portion of the complexes could then permit nickel
ions to enter followed by UreD and UreF dissociation from holourease. I propose that
UreF is the key component involved in precluding nickel ions from binding to the active
site until the carbamylated lysine metallocenter is formed. I suspect that UreF ftmctions
within complexes rather than as the free protein.

Since UreG is produced in large quantities in the cell and it contains a nucleotide-
binding motif typically observed in nucleotide binding proteins, I attempted to purify and
characterize UreG. I hypothesized that UreG may assist in CO2 delivery to the active site
in an energy-dependent manner. Purified UreG did not bind or hydrolyze nucleotides,
contain biotin, nor exhibit carbonic anhydrase activity. Mutations in the UreG P-loop
motif do, however, eliminate detectable urease activity in viva when cells were gown in
the presence of nickel. These results support work done by Mann Lee in which he
identified an in viva energy requirement for urease activity. Although UreG did not bind
or hydrolyze nucleotides, I suspected that UreG may only exhibit nucleotide binding and
hydrolysis activities when complexed with other proteins (see below).

In addition to characterizing UreG, I identified a complex containing UreD, UreF,

and UreG (DF G) in urease deletion mutants. The partially purified complex bormd to an

154
ATP-linked agarose resin. Formation of DFG was not affected by mutations in the UreG

P-loop motif; however, this species was incapable of binding to an ATP-linked agarose
resin. I propose UreG is unable to bind and hydrolyze nucleotides unless complexed with
other accessory proteins. DFG may modulate the ability of UreG to bind and hydrolyze
nucleotides. UreG may act as a molecular switch where the binding and hydrolysis of
nucleotide triphosphate to nucleotide diphosphate triggers the on and off states of UreG.
UreD and UreF may function as nucleotide release proteins, similar to guanine nucleotide
release proteins, or nucleotide hydrolysis activating proteins such as GTPase activating
proteins observed in eukaryotes (1). UreD and UreF in the DFG complex may regulate
metallocenter assembly by modulating the relative concentration of active UreG (NTP-
bound) and inactive form (NDP-bound) in the cell (Fig. 1). An important experiment will
be to test DFG for NTP binding and hydrolysis, but also add NTP to DFG and look for
dissociation of UreD or UreF from the complex. Once NTP is bound to UreG, the protein
functioning as the nucleotide release protein may dissociate. It is also possible that the
proteins only dissociate from UreG after interaction with Apo (i.e., in the DFG-Apo
complexes).

In an attempt to characterize DFG-Apo, I partially purified the complexes in an
Escherichia coli strain that overexpressed ureD and ureF as well as the other urease
genes. These complexes were distinct from those identified in viva previously by Il-Seon
Park. The complexes formed a large aggegate that migated very slowly during
nondenaturing gel electrophoretic analysis. The activation competency, bicarbonate and

nickel requirements, and metal inactivation studies were similar to those observed for D-

155

Apo. Not only were these complexes distinct from those observed previously,
characterization of these complexes did not provide any insight into the possible role of
this species. I conclude that the complex I characterized is not relevant to the in viva
urease activation pathway and results from nonspecific protein interactions probably due
to the overexpression of ureF in the cell. However, in vitro studies involving the
incubation Apo and DFG together did provide some encouraging results. It appeared that
DFG-Apo was formed and its band pattern on a nondenaturing gel was similar to the
DFG-Apo complexes that were observed in viva. This procedure might be used to form
enough DFG-Apo so that NTP binding and hydrolysis studies can be performed.

By combining studies done by me and by other members in the lab, a pathway
describing in viva urease metallocenter assembly has been proposed (Fig. 2; also depicted
in Chapter 1). Nickel ions enter the cell by utilization of a nickel or less selective metal
ion transport system. UreE (CED) binds 6 nickel per dimer and is thought to donate nickel
ions to Apo present in the DFG-Apo species. Formation of DFG-Apo is thought to arise
from sequential binding of UreD, UreF, and UreG to Apo or from binding of the pre-
formed DF G complex to Apo. Interaction of DFG-Apo with UreE results in insertion of
nickel into the active site along with CO2. Interaction of DFG-Apo and UreE has not yet
been detected so this aspect of the model still needs to be tested. Nucleotide hydrolysis is
probably involved at this stage, but it is unknown whether the energy requirement is for
UreE nickel donation, C02 delivery, or removal of an incorrectly bound metal from Apo.

The CO2 delivery system for this process is unknown but accessory proteins are most

 

156

likely involved. Once nickel and CO2 have been successfully inserted into the active site,
the accessory proteins dissociate from urease and functional holoprotein is formed.
Important questions need to be addressed in order to better understand urease
metallocenter assembly. The DF G complex needs to be more fully characterized
including testing for CO2 binding, carbonic anhydrase activity, and K.,, determination for
binding nucleotide using equilibrium dialysis methods. The DFG-Apo observed in viva
needs to be characterized. Does UreG have NTP hydrolysis capabilities in this species
and does this complex directly interact with UreE? Another key question is how CO2
gets into the active site. Are accessory proteins involved or does it simply diffuse into the
active site? Initially I had thought that by purifying the individual accessory proteins and
characterizing them, the roles of these proteins in urease metallocenter assembly would
be easily identified. The difficulties in purification and characterization of UreF and
UreG made me realize that this would not be the best approach. The identification of the
different complexes provided a new direction in my research for understanding the roles
of the accessory proteins. Hopefully my work has helped to shape an in viva urease
metallocenter assembly model that is more detailed and more intriguing then when I

started working on this project.

 

157

15me State Active State
NTP

UreD-UreF-UreG L UreD-UreF-UreG

\em \
"’ NTP
Pr
UreD-UreF-UreG \

NDP
Inactive State

Figure 1. Proposed nucleotide hydrolysis scheme for the DF G complex. The proposed
scheme for the DFG complex involves three different states of UreG: empty, active, and
inactive. It is unclear whether UreD or UreF dissociates from UreG at any of these stages.
The process may only occur if DFG is associated with Apo (i.e., in the DFG-Apo

complexes).

158

Figure 7. Model for in viva urease metallocenter assembly. The incorporation of nickel
into urease apoprotein in viva is proposed to occur by a complex process involving four
accessory proteins: UreD, UreE, UreF, and UreG. Nickel enters the cell using a nickel
transport system. UreE (3) functions as the putative nickel donor and delivers nickel
ions to the DFG-Apo complex. The model proposes that Apo either sequentially binds
UreD, UreF, and UreG, or it binds the DF G complex to form the DFG-Apo complex.
Insertion of nickel and CO2 into the active site may require nucleotide hydrolysis in
which UreG may play a role. Formation of holourease results in dissociation of the

accessory proteins from the enzyme.

 

159

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1 60
REFERENCES

Bourne, H. R., Sanders, D. A. & McCormick, F. (1990). The GTPase
superfamily: a conserved switch for diverse cell functions. Nature 348, 125-132.

 

APPENDIX I

161

 

162

Reprinted (abstracted/excerpted) with permission from Jabri, E., Carr, M. B., Hausinger, R. P. &
Karplus, P. A. (1995). The crystal structure of urease from Klebsiella aerogenes. Science 268.
998-1004, Copyright 1996 American Association for the Advancement of Science.

The Crystal Structure of Urease
from Klebsiella aerogenes

Evelyn Jabri, Mary Beth Carr, Robert P. Hausinger,
P. Andrew Karplus'

The crystal structure of urease from Klebsiella aerogenes has been determined at 2.2 A
resolution and refined to an R factor of 18.2 percent. The enzyme contains four structural
domains: three with novel folds playing structural roles, and an (a (3).; barrel domain. which
contains the bi-nickel center. The two active site nickels are 3.5 A apart. One nickel ion
is coordinated by three ligands (with low occupancy of a fourth ligand) and the second
is coordinated by five ligands. A carbamylated lysine provides an oxygen ligand to each
nickel. explaining why carbon dioxide is required for the activation of urease apoenzyme.
The structure is compatible with a catalytic mechanism whereby urea ligates Nl-1 to
complete its tetrahedral coordination and a hydroxide ligand of Ni-2 attacks the carbonyl
carbon. A surprisingly high structural similarity between the urease catalytic domain and
that of the zinc-dependent adenosine deaminase reveals a remarkable example of active

site divergence.

 

Urczuc (urea amidohydrolasc; EC. 3.5.1.5),
II nickel-dependent metalloenzyme. carn-
lyzcs the hydrolysis of urea to form ammo-
nut and carbon dioxide (I) With a rate
approximately 10” times the rate of the
uncarulyzcd reaction. In 1926, urease was
isolated from seeds of the jack bean plant
(Canavalia ensifamu's) as a pure. crystalline
enzyme by Sumner (2). These crystals. the
first obtained for a known enzyme, played a
decisive role in proving the proteinaceous
nature of enzymes. Approximately 50 years
later, jack bean urease was identified as the
first nickel metallocnzymc (3). Nickel has
since been found to be a component of
hydrogenases for which a crystal structure
was recently reported (4). methyl cocnzymc
M 'reductztscs, and carbon monoxide dehy-
drogenascs (5).

Nickel-dependent ureases have been Iso-
lated from various bacteria. fungi. and high-
er plants (1). Their primary environmental
role is to allow the organism to use cxtcmal
and internally generated urea as at nitrogen
source (6) and, in plants, urcnsc probably
also participates in systemic nitrogen trans-
port pathways and [xrssibly acts as a toxic
dcfcnsc protein (7 ). In agricultural settings,
mpid hydrolysis of fertilizer men by soil
bacterial ureases results in unproductive
volurilizarton of nitrogen and in ammonia
toxicrry or alkaline-Induced plant damage.
Agricultural trials have shown that urease
inhibitors can be Combined with fertilizer to
increase the overall efficiency of nitrogen

 

E.JabruandP,A.KarplusarenrheSoctmolBiocnem-
Istry.M0lecuarandCelBiology.ComeIUrmsrly.
lrhaea,NY.14853.USA MBCanandRP Hausnger
arentheOepanmenlsolMcrobnologyandBIocnem-s-
trybmaanStalo Wy.EastLansing.Ml48824-
it LU

'Towhom correspondenceshoud be directed.

 

utilization (6). Medically. bacterial ureases
are important virulence factors. They are
implicated in the formation of infection-
induced urinary stones (accounting for 15
to 20 percent of all urinary stones). catheter
encrusration. pyelonephritis. and hepatic
encephalopathy (6). The urealyuc Helico-
bacter pylori has also been implicated in
peptic ulceration and possibly stomach can-
cer formation (8). Although some Inhibi-
tors of urease are used in treatment. more
than half of the patients expcricnce adverse
side cffecrs. The commonly used inhibitor.
acetohydroxamic acid. depresses bone mar-
row biosynrhcsis, inhibits DNA synthesis,
and is tcrarogenic in high doses (9).

The best characterized bacterial urease is
that from Klebsiella aerogenes. The native
enzyme has three subunits, a (60.3 kD,
UreC). B (11.7 kD, Uch). and y (11.1 kD,
UreA). reportedly associating with (IrBz'yz)2
stoichiomcrry (10). Jack bean urease. the
benchmark for comparison, exists as a (timer
or hcxamcr of identical 9l-kD subunits (l l ).
Despite the apparent variation in quaternary
structure. amino acid sequence comparison
shows that ureases are homologous. sharing
more than 50 percent sequence identity (1 l.
12). The presence of multiple distinct gene
products related to portions of the jack bean
sequence has also been observed for other
bucrcriul ureases. This clear correspondence
between the sequence of the single subunit
plant unwise and the two or three subunit
bacterial ureases indicates the occurrence of
a gene fusion or disruption events during the
evolution of this enzyme (1). Urease does
not show significant sequence similarity
with other proteins.

The stoichiomerry of nickel and its role
in the catalytic acrivrry of the K. aerogenes
and jack bean ureases have been extensive-

SCIENCE 0 VOL. 268 0 19 MAY 1995

ly studied (1). Stoichiometric analysis of
inhibitor binding to urease (l3) and spec-
troscopic analysis of thiol inhibited urease
(14, 15 ) have shown that both ureases con-
tain a bi-nickel center per active site. This
metallocenter is directly involved in bind-
ing of substrates and inhibitors (16). Recent
evidence has been obtained showrng that
nickel binding to urease requires CO, In-
corporation ptobably lnvolvmg a protein
nucleophile (pK;l > 9.0) (17).

Despite the availability of crystals for
nearly 70 years. a structure of jack bean
urease has not been determined. We have
reproduced the octahedral crystals of jack
bean urease and obtained crystals of K.
aerogenes urease (18). Whereas the jack
bean urease crystals diffract only to ~30 .
those of K. aerogenes urease diffract beyond
2.0 A resolution and can provide a detailed
view of the structure and active site. We
describe here the crystal structure of this
nickel metallocnzyme (urease. from the
bacterium K. aerogenes) at 2.2 resolution.

Structure determination. Crystals of K.
aerogenes urease. selected site-directed mu-
tants (l9). urease apoenzyme (nickel-free)
(20). and selenomethronrne (Se-Met) urc-
ase (21) were grown under the same condi-
tions (18). The phases were determined by
multiple isomorphous replacement (MIR)
at 3.0 resolution with five heavy atom
derivatives and Inclusion of the anomalous
signal (AS) and solvent flattening (SF)
(Table 1). Although connectiviries and side
chain densities were ambiguous in the ini-
tial elecrron density map. the positions of
most of the B strands and a helices were
clear.

An initial Ca trace (720 atoms) from
the MlR-AS-SF minimap was used to build
a polyalanine chain (22) that served as the
starting point for interactive model building
(23). Difference maps for the urease apoen-
zyme and three histidine to alanine mutants
at the active site (His"'“. 1115“”, and
His‘m") (19) provided unambiguous start-
ing points for the insertion of sequence.
Later. the positions of Se-Mct difference
peaks (21 ) and the heavy atom binding sites
served as additional guides for placing se-
quence. Seven rounds of model building.
refinement (24), and phase combination at
3.0 A resolution (25. 26) led to a map in
which the complete polypeptide chains. ex-
cept the last five residues of the 8 subunit,
could be traced unambiguously. Refinement
of this model and phase extension to 2.0
resolution were completed by means of the
simulated annealing and positional refine-
ment protocols of X-PLOR (27). As refine-
mcnr progressed. excess density at the NC
atom of Lys"“7 became stronger and took
on a well-defined branched shape connect-
ing the lysine to both nickel ions (see be-
low). In view of the recently documented

 

 

 

requirement of CO2 for nickel binding ( I7 ).
we modeled this density as a carbamate
derivative of Lyra”. from here on referred
to as Lys"“7'. In the course of refinement.
changes in the density near the active site
led us to suspect that the five crystals
merged in the original native data set
(Natl) (Table 1) might not be perfectly
isomorphous. Difference Fourier analysis
with the unmerged data sets allowed us to
group equivalent data sets on the basis of
the absence (Natl) or presence (Nat3) of
excess active site density. Two structures
were subsequently refined with gradient
minimization in X-PLOR. The Natl struc-
ture. in which a water molecule is bound to
Nl-Z. was refined at 2.2 A resolution with
an R factor of 18.2 percent and an R,“ of
23.2 percent (Table l). The Nat3 structure.
in which an unidentified ligand bridges the
nickel ions. was refined at 2.0 resolution
with an R of 18.5 percent and Rh“. of 22.5
percent. Aside from small differences at the
active site. the Nat2 and Ned structures are
equivalent. The uninterpreted density at
the active site in the Nat3 structure is the
appropriate size for a urea molecule or a
HC03‘. The protein models had good ge-
ometry (28). and most heavy atom positions
were near Cys, Arg. Gln. and Asp residues.
The final electron density map was consis~
tently of high quality (see below) except in
the region of residues r1316 to 01336 where
the density was sparse and the refined mod-
el had high temperature factors.

Overall structure. Klebsiella aerogenes
urease is a tightly associated trirner of
(aB'y)-units in a triangular arrangement
(Fig. IA). This (may); stoichiometry differs
from the proposed (“3272): structure that
was based on the relative intensities of sub-
unit bands in Coomassie blue stained gels
coupled with gel filtration chromatographic
and native gel electrophoretic analysis (IO).
All three subunits make extensive contacts
to build the trirner: each a subunit packs
between the two symmetry related a sub-
units. and contacrs two 8 subunits and two
'y subunits to form the sides of the triangle;
each B subunit packs between two adjacent
(1 subunits at the corners of the triangle;
and each y subunit interacts with two (1
subunits and tightly with two other 7 sub-
units at the crystallographic threefold axis.

Because of the close interactions of the
subunits. it is not obvious which a. B. and
'y chains make up a primary (uBy)-unit.
However. the (aB'y)-unit discussed below
was chosen such that the proximity of the
various termini were compatible with the
known homology of this urease with the
two subunit urease from H. pylori (29) and
the one subunit urease from jack bean (I l ).
The carboxyl terminus of the 'y subunit is
16 from the amino terminus of the 3
subunit. within the distance needed for the

163

insertion of four residues in the H. pylori
urease but requiring a looping out of 30
residues of the jack bean sequence. The
carboxyl terminus of the B subunit is about
35 A from the terminus of the a subunit. a
distance that would readily allow the inscr-

tion of the extra 33 residues that exist in
jack bean urease. The high conservation of
sequence in' all ureases combined with ex-
tensive interactions of the trirner suggest
that all known ureases adopt a similar tri-
meric structure. Approximately 3300 1

Table1.Summaryofcrystallographlcdataandresults. lriterisltydatawerecoiectedatroomtermera-
ture with a San Diego Multlwlre Systems Detector (hardware and software) on a Rigaku RU-200 rotating
anode x-ray generator (47). Heavy atom derivatives were prepwed by soaking crystals at room temper-
ature in crystal storage buffer (100 mM Hepes. pH 7.5. 2.0 M U280.) containing the respective heavy
atom. Forty heavy atom compounds were screened and (Ne derivatives were obtained. Multiple lsomor~
phous replacement phases. at 3.0 A including anomast data (AS). were calcuated by means of the
program Darefl (48). This initial set of phases was mediocre because the heavy atom derivatives sha’ed
common sites and the phasing power beyond ~3.8 A resoiution was weak. The overall figure of merit to
3.0 A resolution was 0.672. The phases were improved with three rounds of SF with zeroing of electron
density at the heavy atom sites to remove heavy atom "ghost" peaks (267. Model building into a 3.0 A
MIRoAS-SF map was done with the programs 0 (22) and CHAIN (23). The phases were improved with
rounds of partial model refinement by means of gradient minimization in the program TNT (24) (33.438
reflections, with weighting to the MlR-AS-SF phases) followed with phase combination by the program
SlGMAAl25). Phaselrnprovementwasevldenced bylmprovedseperationofthedensityoithenlckellone
mdtnprwedcumectMtyandsidechahdensitieshmglmsmthcudedhmnndd. Uponcompletlon
oi the chain tracing. refinement was-continued with x-PLOR (27). The models for N8l2. Nata. and
apoenzymewerealsorefinedhX-PLOR.mefhalmodelsforNat2andNatathde787resldueeand
twoNi‘“ ions. memodellorueaeeapoenzymecontalna 787Wbutbckathetwou’t lonsand
the 002 modification of Lyr’".

 

 

 

 

 

 

 

 

 

Heeobtion Reflections
Data set R '
No. of Correlate '1'“
1A) was Total um (96)
Data colecdon sfatistim
Nat1t 2.0 5 883.385 58.483 93 9.5
Nat2 2.2 2 331.399 42.583 92 11.0
Nata 2.0 3 388.731 58.334 93 8.9
Apoenzyme 2 8 85.114 20.532 95 12.2
HOHgCoH.OO,Ne 3 3 18.880 11.027 89 7.5
EuC 3.3 18.953 12.210 89 8.5
H92( 000), 2.5 87.132 28.709 94 7.8
C(HgOOCCHg, 2.4 89.997 29.872 98 8.5
(CHslaPtXCHfiOO) 2.4 108.954 23.488 98 7.9
SeoMet 3.0 92.935 20.332 90 14.7
Soaking R l I Phaehg power vs
Derivatives cor dltioiis versus Heevyatom
Cone. Time "(3° 3"“ ee—B 8-4 4.3
(mM) (days) A A A
Hteshg statistics
HOi-igC,H.CO,Na 1 5 19.5 e.b 1 .30 1.18 0.79
E 10 7 9.5 a.b 1.48 1.31 0.74
Hg,( H3000 Sat'd. 2 15.3 a.b.c.d.e 1.30 1.18 0.98
C(H . Sat'd. 1 18.3 a.b.c.f.g.h.l.l.k 2.00 1.58 0.97
(cnafucmoool 10 a 12.5 b.g.l.m.n 1.75 1.49 0.97
POM! - - - - 0.825 0.789 0.812
De Ra an . Non- S ' | rms deviations
is set lotion
(A) tione w) gen 3:“ Fl 8...? Bonds Angles
atoms (Al (009)
Refinement statistics
Net2 10-2.2 41.809 5.984 177 18.2 23.2 0.008 1.88
Nata . 10-2.0 55.572 8.002 215 18.5 22.5 0.008 1.98
Apoenzyme 10-2.8 20.184 5.944 157 18.4 25.7 0.009 1.88

 

'Rm-)3ll-(DI/'2(D.Mierelislheinlegraledintensilyofedvenrel|ectlon. Mt-mmwum
analysis. tMprWrmWWrwmmevnwdummwmmwe
andthelackofclostnerror. iFOMlsthemeenIgtnolment(coeheoltheestvnetedprueermn. n-s
lFm-F llxlle.MtareFlsthestructuelector. 18,.lsthecroaemuonnlactarcorruitedlortmmt
setolreuaomtsbcmdnntotaomflchnmtedhmmm

SCIENCE . VOL 268 . 19 MAY l995 90'

 

(10 percent) of the a, B, and y surfaces are
buried to form the (aflvl-unit, and 19 100
A: (23 percent) of the (aBy)- unit surface' Is
buried when t e trimer is in format' t.Ion
The (uB-y)- unit itself forms a T-ahlfped
molecule with dimensions of 75 byBO by 80
A (Fig. 13). The urease (uB‘Yl- unit consists
of four structural domains, two in the a

The structures oi the moose (u trirner end
the (amiunit, (A) A ribbon diagram at the (may)a
tnmer oi urease, as viewed down the crystallographic

u 1)-unit In violet. one In
thte, and the third colored according to the Inde-
ual subunits: u (red). 8 (orange). and 'y (yellow), The
nIckei Ions at each active site are shown as cyan
spheres. The overall shape oi the trImar is triangular
With approximate dimensions oi 110 by 1 10 by 80 A.
The nickel centers are located approximately 50 A
. t c » . . n l I.

(a) A stereo ribbon diagram highlighting the gecond-
‘ ' ‘- l n ll’nf‘l‘l ‘l

 

(orange),andy(yellow)oiurease Thls(uB-y)- )-wunit
chosen such hthat the proximity of the termini corre-
d with equivalent residues In the esubunIt
urease irom )ack been a nlcke l ions (cyan) are
located at the carboxyl termini of the strands In the
But {6)

 

lhe urease subunits showrng the l0ur structural do-
I . . I,

strands (black arrows) are generally numbered in the
order In which they appear In the individual domains
n oi the u
d 5 H6 denot-
barrel excursions. and asterisks (‘) denoting
the residues in domain 2 oi the a subunit This domain

. .4 t I ,I t. .

.
termini oi the a subunit, Extensive hydrophoblc Inter-
actions In the middle 0 t e canyon. and hydrogen
bonds between strand 5‘ and 6' and strands In both
th

that thus In an “Independently folding domain In do—

oithe subunit CIroles represent the mentions
H‘sulw‘ L In?” Hisniufl I

bIndlng

2 to distinguish them lrom the
slrandfi.“

3

1000

 

ary ructure In central six strands
Also In the B suburut a hyphen () )indicates that strand 4

164

chain and one each in the Band 'y chains
(Fig l, nd.C) Three of these domains
appear to Bbe unusual types of folds (30).
They do not contribute any residues to the
active site, but rather have structural roles.
e a subunitl has an (1|?!)a barrel domain
and a primaril yB domain (F‘ 15 g. l).
01.43)

The
barrel is yrather elliptical with the

long axis (about 20 A) connecting strands 3
nd 7. Two extra parallel strand: (9 and 10)
extend the lower portion of strand: 1 to 3.
accentuating the flatten appearance of
e barrel. The active site il located at the
carboxyl termini of the strands, and a hell-
cal excunlon (HZ-H4) between strand 7
and helix 7 form: a "flap" across the active

 

 

 

 

 

 

 

 

 

uku mu- 7 an uvlur w
.4

‘ “mm ISO). and molecular
.1. .I '0.

. An mu _I m.

‘ uuu I HlUlllUlll. MOLSCRIPT (52) and rendered Wil’h Restean ”(’53)

SCIENCE ' VOL 268 - 19 MAY I995

 

site. Two helices (H5 and H6) and a long
loop (residues (1515 to u532) cover the ami-
no-terrnlnal en of the barrel. is loop is
part of an unusual feature in which the
carboxyl terminal 100 raidues wrap com-
pletely around the («(3)ll barrel domain. The
second domain is formed by midues (133
to «129 and r1430 to r1488. Here. eight-
tranded and four-stranded mixed (3 sheets
form the walls of a U-shaped canyon. The
walls are connected by the long strands 5"
and 6" together with strands 10‘ and 11‘.
which go down one side and up the other
The rt: 15 residues of the 8 subunit
fo orm two antiperallel 6 sheets with the
amino-terminal residues of the or subunit

n

jellytomll. The jelllyroll is left- handed
may be the first of this kind observed (3i).
Strand 6 is followed by a long loop that
packs against the loops before and after

strand 3. The last five residues (($102 to
(3106) are disordered. This subunit stabi-
lizes the trirner by associating with domain
2 of its own a subunit and domain 1 of a
symmetry related a subun .

 

165

The 'y subunit adopts a novel a3 domain
with four helices two antiparallel
strands (Fig. 1). Two of the helices (b and
c) and thetwo trands paclt tightly like the
helices of a four-helix bundle and Yhave the
commonly seenn ri-ght handed uownp-d wn-up-
downt opology for that family (32). Helix a
is clearly peripheral and helix d is little
more thn it facilitates
trimer formation through association with

a subunit and the symmetry related y
subunits. The packing of '1 subunits at the
crystallographic threefold axis is dominated
by three copies of helix a. one from each-y
subunit. which peck againste one another' in
an almost orthogonalma

The nickel center. Spectroscopic stud-
ies of jack bean andK aerougenes
have suuested that the two active": site
nickels are within approximately 3. 5 A.
and that each metal ion is approximately
pentacoordinate, with about two imida-
zoles and additional N or O atoms serving
as ligands (l4) Site-directed mutagenesis

.neaeroge s urease tentatively identi-
fied His“ “'1‘ .His"”. and His""6 as three
of the nickel ligands (l9).

 

 

 

F;,¢°endleehownoontouredsi1.5

 

iimeeihermedensiiy. TTiaflgtsewasprodtroedvvithi-WNQ 12$)

zCooscIneiion geome.iryaiihebi-niekelomier Coontiaieaoetncyissooroaa'nareryo.2Aln
itieregionoiihestruoiwe. esestirnaiedbyaumiipioiKQ). ‘

 

 

 

Distance An 1e-
Haul-Mgand . (A) Ligand-netsl-ligand (dist-ll)

N1-l-Lye"”'001 2 .0 Lys""'001~N1-l-H1I""‘N8 92
N1-l-H15"“‘N5 2 .l Lyl"""Wl'Ni-l-HilfluNe 107
N1-1-H1l""7Nl 2 . 2 H1|"““NB~N1'1'H1I"”N£ 95
iii-2 H1:"'“Ns 2.3 H1:"”“Ne-Ni-Z-H1s"""Ne no
li-Z—‘His'm'Ne 2.2 iii-"”‘Ne-N1-2—Lys'm’ 082 88
‘i-Z—Lys‘“ 002 2. 1 Hss'""'N¢-N1~2-Asp"‘°°061 33
-'i-1—Alp"“°061 2.1 H1l"“N¢"N Z—Hat 151
Ni-z—ust-i 2.0 H1:""‘Ne-N1-'2-Lys"” 002 as
H1I“”"N£-N1 Z-Alp""‘°051 88
Ni-l—NLZ 3.5 H1I"”°NE-N1-HZ- -1 98
Alp'“°051-N12-Lys"” 002 163
Alp““"081 Nl-P Hat -1 92

SCIENCE - VOL 268 ' i9 MAY I995

Our structural results agteereeso nably
well with these studies (Fig. 2). The nickel
ions a well ordered (B factors of about
1) and are3.5Aepart. Nl-l is
coordinated by three (two N and one 0)
ligands: Hu‘1“ through the N8 atom.
His"212 (33) through Ne. and Lys“”'
through 001 of the carbamate( (see be-
low). Ni-Z is coordinated byqfive (two N
and three 0) 1113"“; deis"

“through 061.

be thro ugh

Wat-l. and Lys““" through 002 (Table
2). The geometh for Ni- 1 can best be
described as pseud otetrahedral with a
weakly occupied fourth ligand (34) The
geometry for Ni-Z can be descr ibedas
distorted trigonal bipyramidsl or distorted
square pyramidal with His"“ as the api-
cal ligand (Fig.2 and Table 2).

“arose nceof a carbamate ligand to
the nickel‘ ions would have been surprising
if It were not for the recent finding that
activation of urease apoenzyme can
achieved in vitro only in the presence of

1 (17). The activation results and the
structure are consistent with the require-
ment of C02 reacting with'Lys'z'l' prior to
nickel binding. The stnrctu re of the urease

apoenzyme (Table 1) shows no carbamate.
suggesting that carbamylation of Lys““7oc
nreedi iinly aqueous solution. but the
binding of nickel ions is required to stabilise
the carbamylated form. A similar carbamy-
lated lysine has been observed in another
enzyme. namely ribulose- l.hate5-bisphosp
carboxylaseo ygenase (RUBl 5CD) (35)
There .eetbemylatio on is required for the
binding omeg11*. which in turn coordinates
contrutuo reasewh hich
binds metal tignhtly (36). RUBlSCO can
berea lly deactivated by metal chelators.
Although urease can be activated in vitro
by the addition of C01. its activation in
vivo appears to be.aided by specific pro-

teins (

The use of a carbamate ligand. rather
than an Asp or Glu residue. appears to be
necessitated by the position of the active
site in the ((13). barrel in that shorter Asp

lu residues would no t teach the nickel
center. Also. the ability of the carbamateto
rm resonance isomers that allow higher
partial negative charges on the oxygens
oompa to carboxyllc acids may be impor-
tant in the ligation of the nickels or the

Adjacent to the nickel. near Wat-l. is a
pocket roughly the site of urea. which in a
static structure is seques esteted by the side
chain of Cys‘m from a narrow channel
leading to the enzyme surface (Fig.3).
However. the H2-H4 flap (residue «.308 to
0.336). which forms one wall of the chan-
nel. is highly mobile suggesting that this

up may easily open to allow extensive

1N1

access to the active site. In fact. the facile
modification of Cys")l t the base of the
channel by bulky reagents (38) and, in our
analysis, by large heavy atom compounds
such as C(H vOOCCHS)4 suggests that the
channel can readily open.

Th putative urea binding pocket is
lined by residues from loops 1 2. 4. 5. 6, 7.
and 8 of the(aBG).l.yh11rrel (Fig. 3). These
include Al1.1"l6 .iHs '”.Al'1"“‘.
Met‘m“. C s"'l°.G laYnd His “31° Three of
these residues. His““°. Cys‘m”. 11nd His"”o
have been implicated as important by
chemical modification and mutagenesis
studios. Site-directed mutagenesis 11ins““"
to alanine produced an active enzyme with
:1 K111 of l 100 mM compared to 2.3 inM for
wild type (19). This result'suggests that
His"“9 is involved in substmte binding. 11nd
in the structure it is positione 3.1
NM and 3.7 A froin Ni-Z. Furthermore,
His"“° receives a hydrogen hon to N5
from the main chain nitrogen of Asp“"'
and is therefore protunatcd on e.

Modification of Cys‘m” (or its equiva—
lent residue in jack bean urease) by chem-
ical reagent s (Ii. 38) blocks activity.

ixon and colleagues (39) proposed that 11
cysteine serves as 11 general acid in jack
be can urease catalys.L11ter. mutagenesis
studies (36) showyed that is
essennti11l.as 1m alanine mutation of rnhis
residue retain ed 48 percent of t wi d-
type activity while mutations to elarger
residues were more deleterious TheS mph-
yioc coccus xylo sus enzyme does not have 11
cysteine at this position (40C). Sfurther sup-
porting the notion thatC ""9 is not

etn tial for activit in th: structure.
Cys""° S‘y is 4.4 from Wat-l. T11:
effect of larger Cys‘m" mutations could
easily be caused directly by steric effects or

e
Diethylpyrocarbonate (DEP) modifica-
tion of the bacterial urease indicated that .1
histidine with 11 pKno 6.5 is essential for
chitivily( ().4l Site—directed mutagenesis of
produces an enzym at is not
sensitive and although thee mutant has nor~
mai nicke content. it has very low (~0.003
percent) activity (19) These results are
conslsltent with His‘mo actin as 11 catalytic
In the structure. His"J N21 1: 4.8 A
from Ni- 1 11nd 4. 3 A from Wat-1.711: 11K]
and orientation of Hisuno' are influenced by
hydrogen bonds to the 3side chains of
Asp'm’ and Arg“”(‘ (Fig
From the results of biochemical studies.
Dixon 11nd colleagues (39) also proposed

the efficacy of various thiols as inhibitors
an the reactiv 1ty of thiol- specific modifi-
cation rL-11gents for inactivatingK nem genes
urease are consistent with the presence of 11

1002

166

negatively charged group at the active site

once of three“0 acidic residues, Glu
Asp"m Asp" (”an ree main chain car-
bonyl oxy ygens from csiduos Ala“ ”’7.

GI y"277. 11nd Ala""“. The carbonyl oxygens
ofGly‘iz77 11nd Ala‘m“ serve to hold a water
molecule in place. which hydrogen bonds to
both Wat-l and His“”°. in 11 urea-bound
anyme. these carbonyl groups along with
that ofAl11“”’7 11nd possibly 081 of A:sp""so
may be 1nvolved in binding the NH: groups
of urea.

The structure of K. aerogenes urease can
accommodate the mechanistic model pro-
poset by Zemer and colleagues for the jack
bean enzyme (39). The essence of this pro‘
posal is that one nickel ion binds 11 hydrox-
1tle ion. and urea binds to the other nickel.
via O-coordination. to polarize the sub—
strate. Based on the structure. Wat-l would
be the hydroxide and t e urea oxygen
would bind to Ni~l completing its tetrahe-
tlml coordination. Mode ing urea in thins
manner places its oxygen within hydr
bonding distance of of His ”which
may aid in orienting and further polarizing
the carbonyl of urea and account for the
importance of His‘m". The polarized car-
bonyl of urea is subsequently attacked by a
hydroxide. which results in the formation of
11 tetrahedral intermediate. Zemer's model
suggests t at a 11 tracts a proton fro tom

11 water molecule to yield a hydroxide. 061
0sz s‘p ”is in a position to activate Wat- 1

for attack, whereas the putative catalytic

e.iHs “m. is positioned too far from the
attacking hydroxide. For His"no to act as
the cata yticbase. it must move upon bind-
ing of urea to a position favorable for proton
abstraction from Wat- l. The resulting tet~
rahedml intermediate is thought to decom-
pose. wit the participation of a general
acid. to ammonia and 11 Ni- boun carbam-
ate. which subsequently dmociatos. At this
time. the identity of the general acid
present in aerogenes urease (l0) remains

nclen

Although the mechanism outlined
above does account for most of the avail-
able data. the urease structure is a com-
patible with alternative models that may
involve more structural changes at the
active site. For example. urea may displace
Wat-l at Ni-Z and c lnate via the
oxygen. or might even bridge both nickels

' nyl oxygen. in these cu-
es. there are more possible orientations of
bound urea and less can be llld about the
specific roles for active site residues.

A eulloenzyme ve. e
(111(3)a barrel observed in the catalytic Ni-
binding domain of the a subunit 1s a rather
common topology proteins. and active
debate centers on whether the many (118).
barrels that do not significant se-
quence sim ilarityre are result ofconverge nt

or divergent evolution (43). Given thewe lock

with any other protein. we were surprised

 

narrow channel the sideohim
(HIS-11m Lou-315 M319 Alan“ :1" u my A
symmetry

Fig. 3. Slu'eodagramollmactwefslleolmaase. 1119mm
oi‘Cyv‘“

hapodmlroma

.T‘iaohmuielonmdirommidueshlhoamm

reiafitedmolemlemee‘“ Pro'"“°' Them"). mewsitsatthobnooihm

poduomd
suchlhatCBemSyareSIGAlrommeplaneoitheWOlimm ontdyticbou .l'b'mw. Aw“

c11c A 11 am

1:
c1 1.1 Il’. 217
.u-uruu

maaNnrogenata-marebm. oxygenatomsarered. westwatmnmwom. em

(Hi5""'l).y;1 111011101 1‘
101111a0dm1n1museora14A1mprooe

version 2. 35 (BIOS YM).

19 MAY 1995

SCIENCE - VOL. 268 .

TmngureandFlg Mmmmlml

when a structural comparison wit tah
known protein structures (30) revealed that
the (LIB). barrel of the a subunit is strik-
ingly similar to that of adenosine deaminase
(ADA). an enzyme containing one zinc per
active site. is enzym cata yzes t e
deainlnation of adenosine to yield inosine
and ammonia (44). a reaction mechanisti-
cally related to that of urease. h mech-
anisms involve the attack of a metal coor-
dinated water on the amide carbon to form
a tetrahedral intermediate that subsequent-
ly degrades to yield NH; as one of two
products. The nucleophilic water molecule
in ADA is stabilised through hydrogen
bonds with an aspartic acid and with the
catalytic

ADA is one of two members of a new
class of ((113),. barrels characterized by a
unique elliptical barrel axis and a require-
ment for metal ions (43). The second is the
Zn-dependent phosphotriesterase for which
the apoenzyme crystal structure was recent-
ly determined (45) A superposition of ure-
ase and ADA shows that 72 atoms in the
strands of the barrel overlay with l.
root-mean—square (rms) deviation (Fig.
4A). For comparison. the best overlay with
structures of or teh rre presentative members
of the ((13),. barrel family have rms devia-

Fig. 4. oi crease
deeminase (ADA). (A) An may
according“9w to the strategy of Cho
(50The viewis iookingirom romthe arriino to outbox

and adenoeine
the (an). barrels.
Leek

o the ADA active site. Structuraly
equiveim shown (ureaeeADA)
Hie-'3‘:His‘°, Hie"‘°°:His‘7, Aspr'szsp’“.
i-iie“’":Hls”°. HIe““°:Hie2". and Lys""":
”pill,

 

His i7
‘ Ills I36
. ’ 6 His :34
.5” I- Is
295
His 23s 3“
Isa 21:

167

tions of approximatelyA 3.0 A( (46). The
helices in urease aAnd DA all have the
same rilt but are somewhat shifted, so that
90 equivalent residues overlay with .8 A
rms deviation. The active sites of urease and
Aalso show II high degree of structural
similarity (Fig. 4B). The position of the
Ni I- an at-l in urease are structurally
equivalent to the zinc ion and the hydro-
lytic water molecule at the active site of
ADA. Ni 2 ligands His"'" ,Hi?” . and
Asp"’°° of urease are structurally equivalent
to Zn ligands His", His”, and Awni‘ )f
ADA. Lys"z'7' in urease is equivalent to
Asp‘"l . a residue too short to coordinate the
Zn ion. Rather, Hisl” of ADA. structurally
equivalent to NH ligand His"“° of urease.
es 90" relative to His""‘", to take the
place of Lys‘dn' Its a metal ligand. Hls"zn.
.1 NH ligand in urease. is equivalent to

His"“, the proposed cumlytic base in

high degree of global and active site
similarity. combined with pouible mecha-
nistic similarity, strongly suggests that ure-
ase and ADA are homologs. This surprising
homology raises some interesting questions:
how did urease acquire the requirement for
CO, activation, and why is the enzyme
specific for nickel when zinc or other metals
could serve similar functions? Furthermore.

 

"ism
H|s m

SCIENCE 0 VOL. 168 ' i9MAYI995

what mechanisms lie behind the emergence
of (our accessoryp roteins. UreD . UreE
UreF. and UreG (20, 37). which are re-
quired for' In Vivo assemblyof reaseiThus.
in addition to the utility of this stmcture for
inhibitor ign. further study of this system
should lead not only to insight into the
m anism a reguation o urease
also insight into the mechanisms of protein
evolut

REFERENCES AND NOTES
P. 0! Mid (Plerun

m. V.
114. 4324

 

24.

27.

D. E. Tronrud. L F. Ten Eyck. 8. w. Matthews. Acta
Crystalog. A43. 489(1987).The command ile ior

TNTweernodifledtolnciudeaneddtioneiRiactor.

W.,”) calculation beiore iritietion oi each cycle of
refinement. The test date set. generated in X-PLOR.
served as the data set ior Rm calculation whle the
working date set was used in refinement.

. R. J. Read. Acre Oysiabgr. A46. 900 (1990).

SiGMAA weigited structure iectors were used to
generate P.,. am electron densitymepeioruee
In Titthg.

OCP4. "Coleboretive Cornputellonu Proiect. Nun-
ber 4." bid. 050. 760 (1994).

A T. Bn‘nger, A. Krukowsid. J. W. Erickson. Did.
m. 585 (1990).
Alreeiduesarelnthealowedregioneoiueengee
with seven reddues (1.1 percent) ialing In the gen
ermsiyelowedmgionsTwomeintypez'tuns
(Aim. 918””). one is in a type 2 tum (His""’"). two
8'! metal ligands (His"'""‘. Wm”). and one has 3
him 8 iector (Met"°"). Ale"°°‘ hes cieer and wel-
deiined density. The model contains twee cb-pro-
thee: a282, a303, and 0.470.

. C. L Clayton. M. Pollen, H. Kleanthous. B. W. Wren,

S. Tabeochal. MicieicAcidRes. 18. 382 (1990);A.
e. v. Cussac. P. Courcoux. J. Bacterial. 173.
1920(1991).
L HolmendC. Sander. J. Moi. Biol. 233. 123(1990);
N. S. BOutonnel. M. J. Roomm. M.-E. Ochagevie, J.
S. L Wodak. in preparation: D. F. Fischer. H. Woii~
son. 8. L. Un. R. Nussinov. Protein Sci. 3. 7m
(1994). A comprehensive search oi the Protein Data
Bank was done by the three groups cited. The
eeerchbyL. Hoimwr‘ththeprogremoalishowedthet
domeh i oitheasubunit, an(a[3)n barrel Issarrierlo
the («Bin barrel oi adenosine deemlnese. Doman 2
either-subunit. theiseuburit. andtheysubunlthed
nocloceiy eimlu structure.

1W4

88

41.
42.
43.

SCIENCE 0

.J.S.WM.WOM34.

.Theeiectrondensitymapsimsweek

168

167
(1981).

.C.CohmmdD.A.O.Perty.m7.1(1m.

Hie-1" was not studied by site-directed mutagen-
ede because it was not conserved in the Ureaplas-
ma weaiyticum sequence. An analysis oi the ne-
ported U. ureeryiicum sequence (A. Blanchard.
personal communication) indicates that e se-
quencing error caused a reading irame shift so that
about a dozen amino acids. Including Hieez”. re-
ported to be distinct compared to other menses.
are in tact conserved.

dut-
eltybelweenN I endWet-.1 Thedensityis~i.8A
immWet- 1 andthereiore cemotbeesecondweter
rmiecde at it! ocwpemy. We estimate the podtlon
oiWet 1 lshighiyoccupiedasitheeareesonebie
Elector 017.0 A? During the other small portion oi
the time. Wei 1 may instead occupy a position
bridgingthenickeiimeorsoielyaeNi- 1 ligand.

F C. Hertmm and M. R. Hapd. Armfbv M30-
chem. 83 197 (1993).

. P. R. Martin and R. P. Harshger. J. Bot. Chem. 297.

20024 (1992).

.M.B.CerrendR.P.Heuehger.hm1emsol

MeiedooenterAssemory. R. P. Hausinger. G. L Eich-
hom. L. G. Marzilli. Eds. (VCH. New York). in pram.
M.J. Todd and R. P. HassingerJ. Biol. Chem. 296.
24327 (1991).

N. E. Dixon. P. w. nodes. C. Gezzoie. R. L flake-
ley. B. Zemer. Can. J. Biochem. 58. 1335(19w).
J. Jose. 8. Christiane. R. Roeonsteln. F. GOlz. H.
Kdtwasser. FEMS Microbiol. Lett. 80. 277 (1991).
I. S. Parkand R. P. HewhgerJ. Meridian. 12.
51 (1993).

M. J. ToddendR P. M.J. w Oran. 200.
10260 (1991).

G.K.FerberandG.APeteko. 7mm Sol.

VOL. 268 ' 19 MAY I995

46.
47.

48.R.E.Dickereon.

8?

16.228(1m.G.K.Fuber.aIrr.Oph.SYrtaaat
3.409(1993);D.Reudmde.K.Ferber.FASEB
J.,hprm.
O.K.Mson.F.B.W.F.AdeD.m
26212786991).

M. M. W. J. M. Kuo. F. M. Rm. H. M.
Holden. Biodiemlstry33. 150310994).
EJebriandP. AKarplre. mblshed

R.C. Hyatt MethodsEnzymol. 114, 416(1985); N.
H. Xuong. C. Maison. R. Hamh. D. Anderson. J.
Am. Crystaloy. 18. 342 (1985).

J. E.Weinzleri. R.A.Petner.Acta
Oystelogr. B24. 997 (1966). M is a modiied
versimoieproqernmedhttoleboretcryoiQE.

Some.
.P....VLmetlActeOystelog5802(1952).
.WWWNCWW 22.2577

(“383)

M. L. Comoly. Sdence221. 709(1983).

P. Krauls. J. Appt Oystaiiogr. 24. 946 (1991).
DJ. Becmendw.F.Anderson.J.Mot Green a.
219(1968): E. A Merritt and M. E. P. Muphy.Acte
Oystalogr. 050. 869 (1994).

L GiottiaendA Leek. EMBOJ. 4. 823 (1%).

.Wethenki S. PerkmdM. Leeiorselectednutent

andwoenzyrne. R. ShedteiorTAWand

searching

PDBiorhomoiogs. S Eeilckiorlheuseoihlsdetec-
tor. endB. Cerpenterends. Llpperdiorhebiutde-
cuedonoithemeteicenler.SupportedbyUSDA
gent 9303870 (PAK. and R.P.H.) and by NIH train-
hg gent 5T32-GM08384-04 (E.J.). The coorthetee
md slructue iectors we deposited in the Protdn
DeleBerthmtryoodeeiKAUiorMfiKAka
Net3.end3KAUiorthemoenzyme.

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