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This is to certify that me
thesis entitled

A TRIGGER FOR RADICAL GENARATION?
INVESTIGATING THE TERNARY PFL—AE/ADOMET/PFL
COMPLEX

presented by

Ziyang Su

has been accepted towards fulfillment
of the requirements for the

Master of degree in Department of Chemistry
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2/05 01mm ‘

 

A TRIGGER FOR RADICAL GENERATION?
INVESTIGATING THE TERNARY PFL-AE/ADOMET/PF L COMPLEX

By

Ziyang Su

A THESIS

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

MASTER OF SCIENCE
Department of Chemistry

2005

Abstract

A TRIGGER FOR RADICAL GENERATION?
INVESTIGATING THE TERNARY PFL-AE/ADOMET/PFL COMPLEX
By
Ziyang Su

The activation of pyruvate fonnate-lyase (PFL) by its activating enzyme PFL-AE
employs a novel radical mechanism, which utilizes a Fe-S cluster and
S-Adenosylmethionine (SAM or AdoMet) to facilitate the generation of a putative
adenosyl radical. PFL-AE is one representative member of the so-called “Radical
—SAM” superfamily, which is thought to contain more than 600 members, all working
under a common radical mechanism. Previous studies on PFL-AE have indicated
close association of SAM to the Fe-S cluster of PFL-AE, suggesting a pathway from
one sulfide of the cluster to the sulfonium of SAM for the inner sphere electron
transfer. This thesis work focuses on how the substrate PFL interacts with PFL-AE
and the cosubstrate SAM in the ternary PFL-AE/SAM/PFL complex, and how the
pro-S hydrogen atom on glycine 734 of PFL is abstracted by PFL-AE and SAM.
Multiple routes were undertaken to prepare a mutant of PFL, G734A. The activity
assays of PFL(G734A) were performed to study how the mutation would affect the
activation of PFL. The alternative cofactor Te-SAM was also employed to investigate

the ternary complex PFL-AE/Te-SAM/PFL by EPR spectroscopy.

ACKNOWLEDGMENTS

First, I would like to give my warmest thanks to my advisor Prof. Joan Broderick,
who is an adorable scientist and a wonderful advisor. Thank you for being so
generous, innovative and patient. I cannot do this without your guidance and help.

Secondly, I thank the Department of Chemistry at MSU and National Institutes of
Health for financial support in forms of teaching and research assistantships.

Thirdly, I appreciate all the help from my dear group members: Mbako,
Magdalena, Dan, Jeff, Efihalia, Meng, Shujuan, Yi, Egis and Cliff. Especially for
Magdalena, who helped a lot for my thesis writing. Working together with you guys
in the lab has been beautiful memories in my mind.

Also, I would like to thank the great professors in my committee and great friends
I have met in MSU. They have made my life easier and happier.

Finally, I want to give special thanks to my parents, though they are thousands of
miles away, their love is always around to support me. My boyfriend Yiduo supported

me from every aspect and never complained. thank you for always being there for me.

TABLE OF CONTENTS

LIST OF FIGURES ............................................................................ vi

LIST OF SCHEMES .......................................................................... viii
LIST OF TABLES .............................................................................. ix
LIST OF ABBREVIATIONS .................................................................. x
CHAPTER I

INTRODUCTION

1.] Pyruvate formate-lyase ..................................................................... l

[.2 Pyruvate formate-lyase activating enzyme ................................................ 2
1.3 Adenosylmethionine-dependent iron-sulfur enzymes ................................. 4
1.4 Interaction of the [4Fe-48] cluster of PFL-AE with AdoMet .......................... 6
CHAPTER II

EXPRESSION AND CHARACTERIZATION OF PYRUVATE
FORMATE—LYASE (G734A)

Il . I Introduction .............................................................................. 9
[1.2 Experimental methods ................................................................. IO
[1.3 Results and Discussion ................................................................ 13
CHAPTER III

ATTEMPT S TO IMPROVE PREPARATION OF PYRUVATE
F ORMATE-LYASE (G734A)

111.] Introduction ............................................................................ l 8
111.2 Experimental methods ................................................................ l9
["3 Results and Discussion ............................................................... 24
CHAPTER IV

USEAGE OF ALTERNATIVE SUBSTRATE Te-SAM

lV.l Introduction ............................................................................ 30

IV.2 Experimental methods ................................................................ 3|
IV.3 Results and Discussion ............................................................... 33
CHAPTER V

CONCLUSIONS ............................................................................... 36
REFERENCE ................................................................................. 38

Figure 1.1

Figure 1.2

Figure 1.3

Figure [.4

Figure 11.1

Figure 11.2

Figure 11.3

Figure 11.4

Figure 11.5

Figure 11.6

Figure III.1

Figure [11.2

Figure [11.3

Figure [11.4

Figure [11.5

LIST OF FIGURES
Reactions catalyzed by representative members of the radical-SAM
Superfamily ................................................................. 5

Sequence alignment of the Fe-S cluster binding motif in the radical
SAM enzymes ............................................................... 6

Interaction of SAM to the [4Fe-48] cluster of PFL-AE .................. 7

Proposed mechanism for the activation of PF L by PFL-AE and
AdoMet ........................................................................ 8

SDS-PAGE analysis of pKK-PFL (G734A) cells ........................ 15

Chromatogram of purification of pKK-PFL (G734A) by the Anion
Exchange column ......................................................... 15

Chromatogram of purification of pKK-PFL (G734A) by the Gel
Filtration column ............................................................ l6

Elution profile of purification of pKK-PFL (G734A) by the Phenyl
Sepharose column .......................................................... 16

SDS-PAGE analysis of purified pKK-PFL (G734A) off the three
chromatographic columns ................................................... 17

SDS-PAGE analysis of PFL-AE ............................................ l7

Agarose gel of PCR products from pKK-pfl(G734A) with primers
ZS], 282, 283 ................................................................ 26

Agarose gel of PCR products from pKK-pfi(G734A) with primers Z84
and ZSS ..................................................................... 26

SDS-PAGE analysis of pET-30 Ek/LlC-pfi(G734A) cells. . . . ....27

Chromatogram of purification of pET-30 Ek/LlC-pfi (G734A) by the
Ni-HisTrap column ......................................................... 27

SDS-PAGE analysis of pET-3O Ek/LlC-pfl (G734A) off the
Ni-HisTrap column ........................................................ 28

vi

Figure [11.6 SDS-PAGE analysis of pKK-pfi (G734A) in knockout strain KO_pflB
.............................................................................. 30

Figure IV.1 Three degradation pathways of AdoMet .................................. 32

Figure IV.2 X-band EPR spectrum of ternary complex PFL/PFL-AE/Te-AdoMe at
12K .......................................................................... 36

Figure IV.3 X-band EPR spectrum of ternary complex PFL/PFL-AE/Te-AdoMe at
60K ............................................................................ 36

Figure IV.4 Blow-up of Figure IV.3 at the signal site. Details of the doublet
splitting can seen from field 3200 Gauss to 3500 Gauss ................ 37

vii

LIST OF SCHEMES

Scheme [.1 PFL catalyzed interconversion of pyruvate and CoA to acetyl-CoA and

formate ............................................................................ 1
Scheme 1.2 AdoMet-dependent PFL glycyl radical generation by PFL-AE ............. 2
Scheme 11.] Acitvity assay of PFL ........................................................... l3

viii

LIST OF TABLES
Table 111.] Sequence of primers used in cloning ........................................... 20

Table [11.2 Solutions for making competent cells .......................................... 25

ix

LIST OF ABBREVIATIONS
AdoMet/SAM: S—Adenosyl-L-methionine
EPR: Electron paramagnetic resonance
ENDOR: Electron-nuclear double resonance
LB: Luria-Bertani
HEPES: 4-(2-Hydroxyethyl)piperazine- l -ethanesu|fonic acid
PMSF: Phenylmethanesulfonyl fluoride
DTT: Dithiothreitol
SDS-PAGE: Sodium dodecyl sulphate polyacrylamide gel electrophoresis
Tris: Tris-(hydroxymethyl) aminomethane
IPTG: Isopropyl B—D-l-thiogalactopyranoside
Ni-NTA: Nickel-nitrilom'acetic acid agarose
UV-vis: Ultraviolet-visible
OD: Optical density
kDa: kiloDaIton

Kbp: Kilo base pairs

Chapter I

INTRODUCTION

1.1 Pyruvate formate-lyase
The enzyme pyruvate formate-lyase (PFL) catalyzes the reversible conversion of
pyruvate and coenzyme A (CoA) to formate and acetyl-CoA, which is the first

committed step in anaerobic glucose metabolism in E.coli (Scheme 1.1)." 2 PFL has

0

0' PFL
+ HSCoA —> +
H3C 0'

Scheme 1.] PFL catalyzed interconversion of pyruvate and CoA
to acetyI-CoA and formate

the distinction of being the first enzyme identified to contain a stable and catalytically
essential glycyl radical.3‘ 4 This remarkable discovery was made using electron
paramagnetic resonance (EPR) spectroscopy in combination with isotopic labeling,
together with analysis of the products of oxygenolytic cleavage of the
radical-containing protein. This discovery brought up two key questions: first, what is
the mechanism by which a glycyl radical mediates the C-C bond cleavage of pyruvate;
second, how is the glycyl radical generated? The first question has been the subject of
active investigation for nearly 30 years. It is known that the glycyl radical does not
directly participate in catalysis, but rather serves as a source of an unpaired electron
that can be relayed to the active site in the form of a cysteinyl-thiyl radical, which is

directly involved in homolytic cleavage of the pyruvate C-C bond.5' 6 Uncertainty

remains regarding the precise role of the glycyl radical in catalysis. This thesis is a
part of the integrated effort to answer the second question, how the glycyl radical is
generated in pyruvate formate-lyase.

PFL from E. coli is a homodimeric protein of 170 kDa. Early work demonstrated
that PF L was activated under anaerobic conditions by an activating enzyme, in the
presence of S-adenosylmethionine, Fe(11), and reduced flavodoxin as a reductant.7‘ 8
Subsequent work identified the glycyl radical was located on residue G734 of PFL.L3
Concomitant with generation of the glycyl radical, S-adenosylmethionine was cleaved

to produce methionine and S’-deoxyadenosine (Scheme 1.2). Label transfer studies

H
:2: Ad

Ad *H

      

 

Met +
OH HO OH
H H* K j H
ll, _
PFL-GI , PFL-Gl .
6.

Scheme I.2 AdoMet-dependent PFL glycyl radical generation by PFL-AE

demonstrated that a 5’ hydrogen atom of the 5’-deoxyadenosine product originated
from the G734 residue of PF L.9 The H atom abstraction from G734 was shown to be

stereospecific for the pro-S hydrogen.9

1.2 Pyruvate formate-lyase activating enzyme

Pyruvate formate-lyase activating enzyme (PFL-AE) generates the glycyl radical

at G734 on PFL in the presence of a cosubstrate, AdoMet. PFL-AE was initially
isolated aerobically from Ecoli and found to be a 28 kDa monomer with a broad
absorbance from 310 to 550 nm, suggesting the presence of a covalently bound
cofactor.l The first isolation of PFL-AB in its native state under strictly anaerobic
conditions and identification of the presence of an iron-sulfur cluster in PFL-AE were
achieved by Broderick and coworkersw’ ” It was also demonstrated that the Fe-S
cluster of PFL-AE is required for enzymatic activity. A complete description of the
states of the cluster present in the enzyme was provided by Mossbauer spectroscopy.'2
The major component was the cuboidal [3Fe-4S]+ cluster, accounting for 66% of the
total iron, followed by pro-2812* (12% of the total Fe), [4Fe-48]2+ (8% of the total Fe)
and linear [3Fe-4S]+ (~10% of the total Fe). When PFL-AE was anaerobically
reduced with dithionite, complete conversion of all cluster types to [4Fe-4S]2+’+
clusters was observed by Mossbauer spectroscopy. The [4Fe-4S]'+ was identified to
be the catalytically active cluster by a ‘single turnover’ experiment.l3
Deazariboflavin-mediated photoreduction afforded quantitative reduction of PFL-AE
to the [4Fe-4S]I+ state. Spin quantitation of the EPR spectra showed a 1:1
correspondence between the amount of PFL glycyl radical generated and the amount
of [4Fe-4$]'+ cluster present in PFL-AB prior to addition of PFL and suggested that
the final cluster state is [4Fe-48]2+. This is the first direct quantitative spectroscopic
evidence that the [4Fe-4S]l+ of PFL-AE is the catalytically relevant cluster, and this
cluster provides the electron necessary for the reductive cleavage of AdoMet.

Site-directed mutagenesis studies have identified Cy529, Cys33, Cys36 as cluster

ligands in PFL-AE.l4 In general, a similar cluster-binding motif comprised of only
three cysteines (CXXXCXXC) is a common feature to all of the

Fe-S/SAM-dependent enzymes for which the gene sequence is known.”'9

1.3 Adenosylmethionine—dependent iron-sulfur enzymes

Iron-sulfur clusters are among the most ubiquitous and versatile metal centers
found in biological systems. Electron transfer was the first function recognized;
beyond that, there have been other roles for iron-sulfur clusters identified, including
mediating redox and non-redox catalysis, and gene regulation. Aconitase is an enzyme
that utilizes a site-differentiated [4Fe-4S] cluster as a Lewis acid to interconvert
citrate and isocitrate.20 Iron-responsive protein (1RP),2' fumarate-nitrate reduction
protein (FNR),22 SoxR23 utilize iron-sulfur clusters to respond to changes in levels of
iron, oxygen and superoxide respectively.

More recently, a new role for Fe-S cluster has emerged as a number of enzymes
has been identified that utilize Fe-S clusters and AdoMet to initiate radical catalysis.
This Fe-S cluster-mediated radical catalysis includes the generation of catalytically

3. 10, 24, 25

essential glycyl radicals, the generation of substrate radical intermediates,26

cofactor biosynthesis,”29

and repair of DNA damage.30 This novel protein family was
first identified in 2001 and is thought to contain more than 600 members, and was
named the “Radical-SAM Superfamily”.31 Some of the reactions catalyzed by

members of this superfamily are shown in Figure 1.1. Despite the

remarkable diversity of the reactions catalyzed by these enzymes, it is currently

H
”a.
'- 13-01 : E-Gl ,
y PFL-AE ’ ° . 555d

 

 

’H3N

 

 

HNJKNH HN
Meow BioB
H3C
3 COOH

Sugar
H

 

 

 

Phosphate
H
Sugar

 

 

OH OH
H+ + HCOO'

N = adenine. guanine, cytosine, uracil

Figure [.1 Reactions catalyzed by representative members of the radical-SAM superfamily.

PFL-AB, pyruvate formate-lyase activating enzyme; LipA, lipoate synthase; LAM, lysine
2,3-aminomutase; BioB, biotin synthase; SPL, spore photoproduct lyase; ARR, anaerobic

ribonucleotide reductase.

thought that all of these reactions, as well as those radical-SAM reactions yet

undiscovered, involve a common mechanism. A three cysteine CXXXCXXC cluster

binding motif is conserved in all members of the superfamily (Figure 1.2); this

suggests a unique Fe site in the [4Fe-48] cluster, which has been indicated to interact
with AdoMet mediating reductive cleavage of SC bond and radical generation.”35

The work described here focuses on the glycyl radical generation of PFL by PFL-AE

in the presence of AdoMet.

SP Lyase 8 6 IPFATGCMGHCHYCYLQTT
PFL-AE 2 4 ITFFQGCLMRCLYCHNRDT
aRNR-AE 2 O VL FVTGCLHKCEGCYNRST
Biotin Synthase 47 SIKTGACPQDCKYCPQTSR
Lipoate Synthase 48 MILGAICTRRCPFCDVAHG
LAM 1 32 LLITDMCSMYCRHCTRRRF

Figure 1.2 Sequence alignment of the Fe-S cluster-binding motif in the radical SAM enzymes

1.4 Interaction of the [4Fe-48] cluster of PFL-AE with AdoMet

Electron-nuclear double resonance (ENDOR), which probes coupling between
electronic and nuclear spins,36 has been used to define the interaction of AdoMet with
the [4Fe-4S] cluster of PFL-AE. Isotopically labeled S-adenosylmethionines were
prepared with '70 at the carboxylate oxygen, with 13C at the carboxylate carbon, and
with l5N at the amine nitrogen.35 The observation of ENDOR signals for all three
nuclei demonstrated for the first time that AdoMet forms a classical five-membered
chelate ring with the unique iron of the [4Fe-4S] cluster of PFL-AE (Figure 1.5).35
Field-dependence of the 13C, 17O, and 15N ENDOR data further suggests that the
unique iron serves as an anchor to hold the cosubstrate AdoMet in place for reaction.37
Furthermore, ENDOR of methyl-zH-AdoMet and methyl-”C-AdoMet reveals local
orbital overlap of AdoMet with the [4Fe-4S] cluster.34 It was proposed that this orbital

overlap occurred via interaction of one of the rig-bridging sulfides of the [4Fe-4S]+

cluster with the sulfonium of SAM (Figure 1.3); this close contact provides a pathway
for the potential inner-sphere electron transfer from the cluster to AdoMet, which is

expected to facilitate homolytic bond cleavage and creation of the adenosyl radical.”

 

Figure 1.3 Interaction of SAM to the [4Fe-48] cluster of PF L-AE

35 On the basis of the above observations and discussion, a mechanism for the
Fe-S/AdoMet-driven radical chemistry catalyzed by PFL-AE was proposed (Figure
1.4) by our group.38

EPR and ENDOR results provide significant insight into the mechanism by which
PFL is activated by PFL-AE in the presence of AdoMet, yet many questions remain
unresolved here: How does PFL-AE recognize the gly-734 residue in pyruvate
formate-lyase? How do PFL, AdoMet and the iron-sulfur cluster of PF L-AE interact
so as to trigger inner sphere electron transfer and S-C bond cleavage? How does the
5’-deoxyladenosyl radical abstract the pro-S hydrogen from the glycine residue of
PFL? Concerns about the thermodynamic aspects of the radical catalysis remain as
well, such as the dissociation constant of the interaction between PFL and PFL-AE,

and between AdoMet and PFL-AE.

 

CLSS-S \ "§/ e' CLSSSV’ \gg/J
’ I I I ’I
l‘Fe 8 H2 k ’ I‘Fef's H2
Fe’ls I .-~“‘N Fe 5 ll: .~\“\
8.
Cys S/ \s/ ‘0 Cys-S/ \s/ ‘0
o o
[4Fe-4S]2+-AdoMet I4Fe-4SI‘*-AdoMet
A

/ Met + dAdo
\ AdoMet ¢

 

 

 

Ado AdO
’ PFL-Gly PFL-Gly H20,
Cys-S,’ H3C H3O Cyss‘S/g’ H3O .
Cys—S I’Fe \S ‘k / Cys—S Fe \S
’ ’ \
execs H. sacs H.
é I \\\\N e/ Fes\ lFe .\“\\N
/Fe< )Fe“ Sl/Fe \ )F
Cys-S S ‘0 CYS-S S 9‘0
O O
[4Fe-48]2+-Met + dAdo - [4Fe-4S]2+-Met + dAdo -

Figure [.4 proposed mechanism for the activation of PFL by PFL- AE and AdoMet.
Inner sphere electron transfer from a bridging sulfide of the [4Fe-4S]l+ cluster to the
sulfonium of AdoMet causes 0 S bond homolysis, which produces a
5’-deoxyadenosyl radical and methionine. The adenosyl radical abstracts a hydrogen
from Gly734 of PFL and 5’-deoxyadenosine and methionine are replaced with another
AdoMet. The source of the electron is proposed to be a reduced flavodoxin

To address the question how PFL-AE recognizes the eg-734 residue in FF L, a
series of peptides homologous to the gly-734 site of PFL were synthesized and
tested.9 Among them, a heptamer peptide with eg-734 replaced by L-alanine totally
abolished the H atom abstraction by PF L-AE. This suggested that it is very likely a
mutant protein of PFL G734A would be inert as well in terms of radical activation.

One goal of this work is to address the substrate recognition properties of PFL-AE. A

mutant PFL(G734A) was cloned and purified to test its activity as a substrate for

PF L-AE.

Another goal of this project is to investigate the ternary complex
PF L-AE/AdoMet/PFL by ENDOR spectroscopy. The spectroscopic results obtained
so far are restricted to the binary complex PFL-AE/AdoMet, in the absence of the
substrate PFL. Since the binary complex is not active and does not generate a radical,
some important structural/electronic changes must occur upon adding PFL. Either
AdoMet or PFL will be isotopically labeled for ENDOR purpose. In case of mutant
PFL(G734A), if no turnover occurs, ENDOR of sample PFL-AE/labeled
AdoMet/PFL(G734A) could detect the interaction between the [4Fe-4S]l+ cluster of
PFL-AE and AdoMet in the presence of PFL; on the other hand, sample
PF L-AE/AdoMet/Iabeled PFL(G734A) can provide information about the interaction
between the cluster and the substrate PFL. Furthermore, sample with wild type PFL
can be probed by ENDOR via labeled PFL. In this case, the ternary complex
PFL-AE/labeled AdoMet/PFL would generate a glycyl radical and the [4Fe-4S]l+
cluster would be oxidized to [4Fe-4S]2+ (EPR silent). Subsequently ENDOR could
detect the interaction between AdoMet and the glycyl radical. Taken together, study of

the ternary complex would allow a full analysis of the radical generation process.

CHAPTER II
EXPRESSION AND CHARACTERIZATION OF PYRUVATE

FORMATE-LYASE (G734A)

11.1 Introduction

One key question of the radical generation mechanism is how PFL-AE
recognized the glycine 734 as the active site in pyruvate fomate-lyase. In attempts to
investigate the substrate recognition properties of PFL-AE, a series of peptides
homologous to the active site of PFL were synthesized and tested for substrate
efficiency.9 Among them, a heptamer peptide with glycine-734 replaced by L-alanine
totally abolished the H atom abstraction by PFL-AE. It would be interesting to study
the mutant of PFL, replacing glycine at 734 by L-alanine, to determine if it can act as
a substrate for PFL-AE.

If PFL (G734A) cannot act as a substrate as expected, ENDOR spectroscopy can
be performed on various PFL-AE/AdoMet/ PF L (G734A) ternary complexes. Sample
with labeled AdoMet can help to detect the interaction between [4Fe-4S]+ cluster and
AdoMet in presence of PFL (G734A); sample with labeled PFL (G734A) can
facilitate detection of the interaction between [4Fe-4S]+ and PF L (G734A), which can
not be obtained from binary complex PFL-AE/AdoMet. At the same time, EPR

spectroscopy will be employed to help monitor the oxidation state and cluster type of

PFL-AE.

10

11.2 Experimental methods
11.2.1 Materials

The plasmids pKK-PF L and pMG-AE were obtained as a generous gift from John
Kozarich (Merck). S-Deazariboflavin was synthesized in our laboratory according to

39, 40

published procedures, and characterized using NMR and Mass Spectrometry. All

other chemicals were obtained commercially and used as received.

11.2.2 Growth and expression of PF L (G734A)

The mutated PFL (G734A) gene was subcloned into the plasmid pKK-PFL.
PKK-PFL (G734A) was then used to transform E. coli strain BL21(DE3)pLysS (from
Novagene). A single colony of transformed cells was used to inoculate 50 mL LB
media containing SOug/mL ampicillin (LB/Amp). This culture was grown for 16h to
saturation and then used to inoculate LB/Amp in 6 flasks containing 800 mL media
each. The culture was then allowed to grow for 24 h at 37°C in shaker at 250 rpm
before being harvested by centrifugation at 8,000 rpm for 15 min (Sorvall GS3 rotor).

The supernatant was decanted and the cells fieezed at -80°C.

11.2.3 Purification of PFL (G734A)

PF L (G734A) was purified from the BL21(DE3) pLysS/pKK-PFL(G734A) cells.
Cell paste (typically 10-15 g) was suspended in enzymatic lysis buffer (5 mL per gram
of cell paste) containing 20 mM Hepes (pH 7.2), 1% (w/v) Triton X-100, 5% (w/v)
glycerol, 10 mM MgC12, 8 mg lysozyme, 1 mM PMSF, and trace amounts
(approximately 0.1mg each) RNase A and DNase I. The suspension was agitated and
then incubated at ambient temperature for l h. The lysed cells were centrifuged at

15,000 rpm for 20 min at 4°C.

11

The crude extract was purified by fast protein liquid chromatography (F PLC) in
three steps: first, the crude extract (approximately 50 mL) was loaded onto an Accell
Plus QMA Anion Exchange column equilibrated with buffer A (20 mM Hepes, lmM
DTI‘, pH 7.2). The column was washed with 300 mL of buffer A prior to running a
gradient from buffer A to buffer B (20 mM Hepes, 500 mM NaCl, 1 mM DTT, pH 7.2)
over 900 mL. PFL eluted at approximately 250 mM NaCl. Fractions containing >
70% pure PFL (as judged by SDS-PAGE on a 12% Tris-HCI gel) were combined,
flash frozen and stored at -80°C. The second step was to load fractions from the ion
exchange column onto a gel filtration column (Superose 12 10/300 GL, Amersham
Bioscience) with buffer A, PFL eluted approximately after 40 min (1 mL/min).
Fractions containing > 75% pure PFL were combined and dialyzed against buffer C
(40 mM Hepes, pH 7.2, 1 M ammonium sulfate, 1 mM DTT), and centrifuged to
remove precipitated protein. Thirdly, the supernatant was loaded onto a hydrophobic
interaction column (Phenyl-Sepharose, Pharmacia 16/10) equilibrated with buffer C.
The column was washed with 50 mL of buffer C prior to running a gradient from
buffer C to buffer A over 50 mL, followed by a wash with 50 mL of buffer A. PFL
was eluted through the last half of the gradient. Fractions containing >80% pure PFL

were combined, concentrated, flash-frozen and stored at -80°C.

11.2.4 Preparation of PFL-AE
The plasmid pCAL-n-AE3 (from Stratagene) was transformed into E.coll'

BL21(DE3)pLysS strain. PFL-AE was overexpressed and purified as described in the

published methods.” 3“

11.2.5 Protein assays

12

Routine determinations of proteins concentrations were done by the method of
Bradford,“ using a kit purchased from Bio-Rad, and bovine serum albumin as a

standard.

11.2.6 Activity assay of PFL (G734A)

Scheme 1 illustrates how the activity assay was performed. Under strict anaerobic
conditions (02 level < lppm), the PFL-AE reaction mix was made to a final volume
of 450 1.1L: 0.10 M Tris chloride, pH 8.1, 0.15 M NaCl, 10 mM oxamate, 8 mM DTT,
0.6 uM PFL-AE, 50 M PFL,, 0.2 mM AdoMet, and 0.2 mM 5’-Deazariboflavin
(stock solution dissolved in DMSO). This mix was prepared in the anaerobic chamber
by combining reagents from stock solutions in the order listed to the final
concentrations indicated. In all cases, 5’-deazariboflavin acted as a photoreductant
and was added last in all cases. The samples were then allowed to sit in ambient light
in the chamber for at least 30min, before an aliquot was removed for assay of active

PFL through the coupling assay.

SAM met + 5'dAdoMet

 

 

K / pyruvate
PFL, a PFLa CoA
AE, DTTJBdtioed flavodoxin
\‘ formate
ll
CoA memo“ NADH NAD+

citrate «y oxaloacetate AJ—malate

citrate synthase malic dehydrogenase

Scheme II.1 Activity assay of PFL

l3

The coupling assay mix contained 0.1 M Tris chloride, pH 8.1, 3 mM NAD+,
55uM CoA, 0.1 mg BSA, 10 mM pyruvate, 10 mM malate, 20 units citrate synthase,
300 units malic dehydrogenase, and 10 mM DTT. All reagents except DTT were
combined and made anaerobic by freeze-pump-thaw cycles. The DTT was added after
bringing the anaerobic mix into the anaerobic chamber. To assay PFL activity, 690uL
of this mix and IOuL of the activated PFL described above were combined in a quartz
cuvette, which was then sealed with a septum and brought out of the anaerobic
chamber to monitor the production of NADH by the increase in absorbance at 340 nm.
1f PFL-AE and AdoMet activate PFL, then PFL, will catalyze the conversion of
pyruvate and CoA to formate and acetyl-CoA. The remaining reactions illustrated in
Scheme 1 couple acetyl-CoA production to NAD reduction, which is monitored by
UV-vis. Therefore, the production of NADH is related to PF L activation. PFL’s
activity was calculated as follows:

U PFL,, (in cuvette) = Rate (A U/sec) x 1/8 (moi/L *Au) x Vol (L) x 60 (sec/min) x 106
(pmoI/mol) Equation 11.1
In the above equation, Rate is the production of NADH, a is the extinction coefficient
of NADH, Vol is the volume of the solution under test in the cuvette. The assay was

repeated every other 20 min until the reading reached saturation.

11. 3 Results and Discussion
11.3.1 Expression and purification of PFL (G734A)

PFL (G734A) was overexpressed from BL21(DE3)pLysS cells harboring the
pKK-PFL plasmid (SDS-PAGE gel see Figure 11. 1). The cells were lysed by an
enzymatic procedure. Crude extract was loaded onto columns of Biological Systems

in the order of ion exchange, gel filtration and hydrophobic interaction

14

(chromatograms see Figure 11.2, 11.3, 11.4, respectively). Fractions off the three

columns yield > 90% pure PFL (SDS-PAGE gel see Figure 11.5).

1 2 3 4 5

 

Figure 11.1 SDS-PAGE analysis of pKK-PFL (G734A) cells. Lane 1, molecular
marker (kDa); lane 2-3, overnight culture; lane 4-5, cells before harvesting

UV abs

100% Buffer B
. —UV
1.2 _ Gradient

0.8-
l
0.6~
0.4-

0.2-l

 

0.0

 

 

0.0 .015 I110 ' 115110 .215 .310 I 31.5—r 410—. 415 v 510
Time (h)

Figure 11.2 Elution profile of purification of pKK-PFL (G734A) for

the Anion Exchange column, PFL eluted at approximately 3.3

hours.

UV abs

 

2.00

1.50 ..

1.00

 

 

 

0.50 T- H I l
0.0 0.5 1.0

Time (h)

Figure [1.3 Elution profile of purification of pKK-PFL (G734A) for
the Gel Filtration column, PFL eluted at approximately 0.6 hours.

 

 

100% Buffer B

 

 

1.4-
‘ ——UV
1.2 — ~ Gradient
1.0d
3 0.8:
m '1
>
3

0.6 -

 

 

 

2.0 2.5
Time (h)

Figure II.4 Elution profile of purification of pKK-PFL (G734A) for
the Phenyl Sepharose column, PFL was eluted through last half of

the gradient.

16

 

Figure 11.5 SDS-PAGE analysis of purified pKK-PFL (G734A) off the three
chromatographic columns. Lane 1, fraction 21; lane 2, fraction 23; lane 3,
molecular marker (kDa); lane 4-6, fraction 24-26. Fractions 23-26 were pooled.

11.3.2 Expression and purification of PFL-AE

PFL-AB was purified from BL21(DE3)pLysS cell harboring the pCAL-n-AE3
expression vector. Proteins off the second run of the gel filtration column showed high
purity (Figure 11.5). The purest fractions were combined, concentrated and stored

under nitrogen in small aliquots at -80°C.

Figure 11.6 SDS-PAGE analysis of
PFL-AB. Lane 1, molecular marker
(kDa); lane 2-4, purified PFL-AB.

 

11.3.3 Activity assay of PFL (G734A)

Activity assays were carried out for the purified PFL(G734A) and isolated
PFL-AE and AdoMet, according to the procedures described previously. As a result,
the activated mutant PF L (G734A) showed specific activity of 0.685 unit/mg, which
is approximately 0.5% of the value obtained for wild type PFL (200 units/mg).9 As
discussed in the introduction part, this mutant G734A was not expected to be an active
substrate for PF L-AE. It is most likely that the activity comes from a small amount of
wild-type PFL, which was coexpressed from the Ecoll‘ strain simultaneously with the
mutant. The wild-type PFL expressed has almost the same size, ion affinity,
hydrophobic affinity as the mutant G734A, thus making it difficult to be removed via
normal purification methods. In order to obtain more pure PFL (G734), alternative

methods need to be explored, which will be discussed in the following Chapter.

18

CHAPTER III
ATTEMPTS TO IMPROVE THE PREPARATION OF PYRUVATE

FORMATE-LYASE (G734A)

111.1 Introduction

The small fraction of activity observed from the activity assay of mutant
PFL(G734A) was assumed to be the result of trace amount of wild-type PFL in the
sample, which is difficult to be removed via normal purification methods. In order to
obtain more pure mutant, two methods have been undertaken: one is to clone PFL
(G734A) into plasmids with an N-terminal Histidine tag to generate PFL
(G734A)‘H156, and purify the protein through metal affinity chromatography. Since
the wild-type PFL does not contain a His-tag, affinity chromatography would allow
separation of the wild type PFL and the mutant. Two plasmids were employed,
pET-28a and pET-30 Ek/LIC. The former requires digestion with the restriction
enzymes; the latter is ligation independent, does not require restriction digestion.

The second method that was chosen to obtain pure G734A PFL mutant was ‘gene
gorging’. The goal of this method was to make precise mutations in the Ecoli genome
at high frequencies that resulted in efficient replacement of the wild type allele with
the modified sequence.42 In this method, a special knockout strain of E.coli was
employed, in which the wild-type PFL gene was deleted. The cloning of PFL (G734A)
into the knockout strain will then allow the production of only mutated PFL protein,
without any wild-type present. The activity assay of the pure PFL mutant should be
able to provide information about how the mutation from glycine to alanine at residue

734 affects the function of PFL.

19

111.2 Experimental methods

111.2.1 Materials

Pfu Turbo polymerase was purchased from Stratagene. Restriction endonucleases
and T4 ligase were purchased from New England Biolabs. Oligonucleotide primers
were synthesized by Integrated DNA Technologies and are listed in Table 111.1.
Plasmids pET-28a, pET-3O Ek/LIC were obtained from Novagene, as were competent
E. Coli Novablue and BL21(DE3)pLysS cells. Nickel-nitrilotriacetic acid agarose
(Ni-NTA) column and Ni-HisTrap column (1 mL) were purchased from Amersham
Biosciences. Ecoli knockout strains KO_pflB were obtained from Nara Institute of
Science and Technology (Nara, Japan). Bradford dye-binding reagent for protein
quantification was obtained from Pierce. All buffers and chemicals used were of the
highest quality available and used as received.

Table [11.1 Sequence of primers used in cloning.

 

 

 

 

 

 

 

 

 

Primer Sequence Purpose

ZS 1 5’-CCGAATTCTCCGAGCTTAA Forward primer for cloning
TGAAAAG-3’ PFL(G734A) into pET28aa

ZSZ 5’-CGCCAAGC 1“1'1'1ACATAGA Reverse primer for cloning
TTGAGTGAA-3’ PFL(G734A) into pET28ab

ZS3 5’-GCCGCCGCTAGCTCCGAGC Forward primer for cloning
TTAATGAAAAG-3’ PFL(G734A) into pET28ac

ZS4 5’-GACGACGACAAGATGTCC Forward primer for cloning PFL
GAGC-3’ (G734A) into pET30 Ek/LICd

ZSS 5’-GAGGAGAAGCCCGG1 1 1A Reverse primer for cloning PFL
CATAGATTG-3’ (G734A) into pET30 Ek/LlCe

 

 

 

 

aUnderlined bases represent EcoRl restriction site
t)Underlined bases represent Hindlll restriction site
cUnderlined bases represent Nhel restriction site

d. . . . .
cUnderllned bases represent requlred overhangs for sense and antlsense prlmers

20

111.2.2 General procedures

The polymerase chain reaction (PCR) was carried out using Dr. Geiger’s
thermocycler, gene amp PCR system 2400 (from Perkin Elmer). Each amplification
reaction contained 20 pmol of each primer, 20 nmol of each deoxynucleoside
tripohsphate, 1 U of Pfu Turbo polymerase, 5 ILL of 10x Pfu Turbo polymerase buffer
and 1-3 pg of DNA [pKK-PFL (G734A)] in a final volume of 50 1.11.. Polymerase was
always added last. Afier a 2 min denaturation step at 95 °C, 30 cycles of the following
program were performed: 15 seconds at 95 °C, 30 seconds at 55 °C, and 90 seconds at
72 °C. The mix was finally cooled down to 4 °C. DNA sequencing was carried out at

the Michigan State University Genomics Technology Support Facility.

111.2.3 Cloning of PF L (G734A) into plasmid pET-28a

The PFL (G734A) gene was amplified from pKK-PFL plasmid by PCR using
primers ZSl/ZSZ and 283/282 (Table 1). Primer 25] contained an EcoRI restriction
site flanked by a 2 base GC clamp and the first 18 bases of the PFL (G734A) gene.
Primer ZSZ contained a HindIII restriction site flanked by a 4 base GC clamp and the
last 18 bases of the PF L (G734A) gene, which included the stop codon. Primer ZS3
contained a Nhel restriction site flanked by a 6 base GC clamp and the first 18 bases
of the PFL (G734A) gene. Two pairs of primers (ZSl/ZS2 and 283/252) were used to
amplify PFL gene by PCR independently. Subsequent to amplification by PCR, the
2283 bp fragment was purified from agarose gel, and digested with EcoRI/Hindlll,

and Nhel/Hindlll, respectively. The digested fragments were ligated into plasmid

21

pET28a that had been similarly digested. All procedures were carried out by standard

methods.43

111.2.4 Cloning of PF L (G734A) into plasmid pET-30 Ek/LIC

Plasmid pET-30 Ek/LIC is a member of Ligation Independent Cloning (LIC)
vectors that do not require digestion with restriction enzymes. pET-30 Ek/LIC kit was
purchased from Novagen, and contained the following components: pET-30 Ek/LIC
vector, T4 DNA polymerase (LIC-qualified), T4 DNA polymerase buffer, dATP,
EDTA, Novablue GigaSingles competent cells, BL21(DE3)pLysS competent cells.
Two primers ZS4 and ZSS (Table l) were synthesized for the purpose of PCR. ZS4
contains required overhangs and the first 7 bases of PFL (G734A) gene. 285 contains
required overhangs and the last 12 bases of PF L (G734A) gene. PCR was performed
according to the general procedures with Pfu Turbo DNA polymerase. After
amplification, the 2283 bp target insert was purified from agarose gel and treated with
T4 DNA polymerase (from the kit). The treated target was subsequently annealed to
the vector pET-30 Ek/LIC and transformed into competent Ecoli cells. All procedures
after the PCR were carried out according to pET Ek/LIC User Protocols.44 The
identity of the PFL (G734A) gene for the plasmid pET-3O Ek/LIC was confirmed by
DNA sequencing, and the resulting plasmid construct was designated pET-30

Ek/LIC-pfl (G734A).

111.2.5 Expression and purification of the pET-30 Ek/LIC-pfl (G734A) gene product

A single colony of E. coli BL21(DE3)pLysS harboring the pET-30 Ek/LIC-pfl

22

(G734A) plasmid was selected from a Luria-Bertani (LB) agar plate supplemented
with 10 ugmL—l of kanamycin, used to inoculate 50 mL of LB media containing 10
ugmL'l of kanamycin, and grown for 12 h at 37 °C with shaking (250 rpm). This
culture was used to inoculate LB/Kan in 6 flasks containing 800 mL media each.
When an ODwo of ~06 was reached, expression of the gene PFL(G734A) was
induced by addition of 1PTG to a final concentration of 1 mM and was allowed to
grow for an additional 3 h. Cells were harvested by centrifugation at 18,000 rpm at 4

°C for 30 min, frozen with liquid nitrogen and stored at -80°C.

Cell paste (typically 10-15 g) was suspended in enzymatic lysis buffer (5 mL per
gram of cell paste) containing 50 mM Hepes, pH 7.5, 300 mM NaCl, 10 mM
imidazole, 1% (w/v) Triton X-100, 5% (w/v) glycerol, 8 mg lysozyme, 1 mM PMSF
and trace amounts (approximately 0.1mg each) RNase A and DNase I. The suspension
was agitated and incubated at ambient temperature for 1 h. The lysed cells were
centrifuged at 15,000 rpm for 20min at 4°C. The crude extract (approximately 25mL)
was loaded onto a Ni-NTA superflow column (Pharmacia, now Amersham Bioscience)
equilibrated with lOOmL loading buffer (50li Hepes, 300mM NaCl, 5mM B-ME,
5% (w/v) glycerol, lOmM imidazole). The column was washed with a step gradient
from loading buffer to elution buffer of high concentration of imidazole (SOOmM,
other components are the same as loading buffer) in 4 steps: 10% of elution buffer
followed by 20%, 50% and 75%, with two column volumes (40 mL) in each step.
PFL (G734A) eluted during the 50% step. Fractions containing >80% proteins were
combined and frozen. A different Nickel column (HisTrap HP, lmL, from Amersham
Bioscience) was also used to purify pET-30 Ek/LIC-pfl (G734A), less amount of

crude extract (approximately 7mL) was loaded onto this column. Loading buffer

23

contains 20 mM Hepes, pH 7.2, lOmM MgClz, 5% glycerol and IOmM Imidazole;
elution buffer contains all the same except 500 mM Imidazole. Similar program was

run for this column as the Ni-NTA column.

111.2.6 Usage of knockout strain of E.coli to prepare PFL (G734A)
Kanamycin-resistant Ecoli knockout strain with the deletion of wild-type
PFL gene was ordered from Japan, denoted KO_pflB. It was obtained as a solid
material and was stored at 4°C. In order to transform PFL (G734A) gene into
KO_pflB, chemically competent cells had to be prepared. Calcium chloride was used
to make competent cells.” 45 Five solutions were prepared, as listed in Table 111.2. All
of the solutions were sterilized by autoclaving. On Day One, 5 mL of LB was
inoculated with a single colony of the strain KO_pflB, kanamycin was added to a
concentration of 10 ug/ml. The media was placed in a shaker at 250 rpm at 37°C
overnight. Day Two in the morning, 100 mL of LB/kan medium was inoculated with
1.0 mL of the overnight culture. While the cells were grown at 37°C, solutions A, B
and C were chilled in an ice bath. Also, a 500 mL centrifuge bottle was filled with
~25% bleach and allowed to stand on the benchtop for approximately 2 h. As soon as
the cells reached OD600 of 0.4-0.6, the centrifuge bottle was rinsed several times with
the sterile H20 prepared earlier. All the following steps were performed in a sterile
hood and cells were kept cold at all times. The cells were transferred to the centrifuge
bottle, harvested in prechilled rotor at 8,000 rpm for 5 min at 4 °C. The medium was
decanted and gently resuspended the cells in solution A, harvested by centrifugation at

8,000 rpm for 5 min at 4 °C. Afier decanting the salt solution, cells were gently

24

resuspended in solution B on ice. Cells were allowed to stand on ice for a minimum of
30 min before being harvested by centrifugation. Afier removal of solution B, cells
were gently resuspended in 4.0 mL of 100 mM CaCl2 / 15% glycerol (solution C).
Aliquots (0.1-0.25 mL) of the competent cells were transferred to sterile, labeled,
chilled microcentrifuge tubes. Each tube was quick-frozen in liquid N2 and then
stored at -80 °C.

The gene pKK-PFL(G734A) was transformed into the competent cells prepared
above. Cells were grown at the same conditions as described in section 111.2.5 to
express PFL(G734A).

Table III.2 Solutions for making competent cells

 

0.9% NaCl, 100 mL

100 mM CaCl2, SOmL

100 mM CaCl2, 15% glycerol (w/v), 100 mL
1.2 L distilled H20

100 mL LB medium (500 mL Erlenmeyer flask)

 

 

 

 

mUOCU>

 

 

 

 

111.3 Results and Discussion
111.3.1 Cloning of PFL(G734A) into plasmid pET-28a

The PFL(G734A) gene was amplified from pKK-PFL plasmid by PCR using
primers ZSl/ZSZ and ZS3/ZS2 (Table 111.1), as judged by agarose gel (Figure 111.1).
Purified gene was digested with EcoRI/Hindlll or NheI/Hindlll, respectively.
Difl'lculties were encountered while trying to perform a double digest of the plasmid
pET-28a with two pairs of enzymes shown above. Multiple attempts were undertaken

to digest the plasmid yet all were unsuccessful. At this point, a ligation independent

25

vector pET-30 Ek/LIC was chosen for cloning.

4.0
3.0
2.0

1.5
1.0

 

Figure [11.1 Agarose gel of PCR products from pKK-pfl(G734A) with primers
ZSl, 282, 283. Lane 1-2, gene amplified by primers ZSl/ZS2; lane 3, molecular
marker (Kbp); lane 4-5, gene amplified by primers ZSZ/ZS3.

111.3.2 Cloning of PFL(G734A) into pET-30 Ek/LIC and expression of the gene
product

The PFL(G734A) gene (2.3 Kbp) was amplified from PCR as judged by agarose
gel (Figure 111.2). The amplified target was then treated with T4 DNA polymerase and
annealed to the vector pET-30 Ek/LIC, leading to a plasmid construct pET-30

Ek/LIC—pfl(G734A). The presence of the mutated PFL gene was confirmed by

1 2 3

 

Figure III.2 Agarose gel of PCR products from pKK-pfl(G734A) with primers
ZS4 and 285. Lane 1-2,PCR products; lane 3, molecular marker (Kbp)

26

sequencing. Proteins overexpressed from BL21(DE3)pLysS cells harboring the

pET-30 Ek/LlC-pfl(G734A) plasmid (SDS-PAGE gel see Figure 111.3).

123456789

    

Figure 111.3 SDS-PAGE analysis of pET-30 Ek/LIC-pfl (G734A)cells
lane 1-4, cell pastes before induction; lane 5, molecular marker (KDa);
lane 6-9, cell pastes 3 hr afier induction with 1PTG

111.3.3 Purification of pET-30 Ek/LIC-pfl(G734A)
Cells were lysed by an enzymatic procedure; crude extract was loaded onto

Ni-NTA affinity column (Figure 111.4). Protein eluted from the column was analyzed

 

 

 

 

51 — 100
_ — UV abs
~ Gradient
4-
- 75
3' m
w . a"
a l . - 50 a;
> . m
:3 2- 39
. ‘\
' / \1 - 25
1- \/\/
0 r I - l ' I ' I ' 0
0 5 10 15 20 25

Volume (ml)
Figure [11.4 Elution profile of purification of pET-30 Ek/LIC-pfl (G734A)
by the Ni-HisTrap column. PFL eluted at approximately 16 min.

27

97.4
66.2

45.0

 

Figure 111.5 SDS—PAGE analysis of pET-30 Ek/LlC-pfl (G734A) fractions off the
Ni His Trap _1mL column. Lane 1-2, 4-5, fractions from the column; lane 3, molecular
maker (kDa).

by SDS-PAGE. The band corresponding to the eluted protein appeared at lower
position (approximately 70 kDa) (Figure [11.5) than expected for a normal size PF L
protein (85 kDa). SDS-PAGE was repeated to make sure that the protein gel was
running properly. In order to investigate this problem, whole cell samples were
examined. It was found that a strong band of expressed PFL at 85 kDa was present in
these samples. However, afier the enzymatic lysis was performed a protein of smaller
molecular mass (70 kDa) was again obtained. This indicated that the enzyme
PF L(G734A) was cleaved during lysis. To prevent protease from cleaving the enzyme,
more cells were grown and lysed in a lysis buffer containing Protease Inhibitor
Cocktail set 111 (from CALBIOCHEM) in addition to PMSF. However, this
modification did not stop the cleavage of PFL(G734A). N-terminal amino acid
sequence of the cleaved sample showed that the first five amino acids of the obtained

protein were different from the ones expected for PFL (MSELN). The sequencing

28

confirmed the cleavage of the PFL protein during lysis and indicated the need for a

different method of obtaining mutated PFL protein.

111.3.4 Knockout strain of E.coli KO_pflB

Chemically competent cells of KO_pflB were prepared as described in the
experimental part. Transformation of the gene pKK-pfl(G734A) into these cells was
performed but it turned out to be inefficient. For majority of the experiments, no
bacterial colonies were obtained on media plates. In a single experiment, a few
colonies were observed from one transformation; and a well isolated colony was used
to inoculate a cell culture. However, no overexpression of the PFL protein was
observed (Figure 111.6). No band present at 85 kDa on the SDS-PAGE gel. Another
protein with mass of 55 kDa was expressed to a certain level. In order to investigate
the best conditions to increase the efficiency of transformation, different amounts of
DNA were used (from 1 to 20 ug), and different heatshock times (between 30 s and 90
s) were applied. But none of these efforts showed any obvious improvement. These
indicated that it is very likely the cells made were not competent enough. Recently,
electroporation has been widely used for transformation procedures. In this method,
an intense electric field is applied to make the cell membranes transiently permeable,
allowing DNA to enter the cell. It is suggested that the electroporation method be used
to make the KO_pflB cells competent, subsequently transform, and express the

mutant PFL(G734A).

29

97.4
66.2

45.0
31.0

 

Figure [11.6 SDS-PAGE analysis of pKK_pfl (G734A) in knock out strain KO_pflB
lane 1,pre-induced cells; lane 2, post-induced cells; lane 3, molecular marker (KDa).

30

CHAPTER IV

USAGE OF ALTERNATIVE SUBSTRATE Te-AdoMet

1V.1 Introduction

Under physiological conditions, the cofactor AdoMet is not stable. It degrades by
three independent processes: the, pH—dependent intramolecular attack of the
a-carboxylate group onto the y-carbon to afford homoserine lactone and
5’-methylthioadenosine (MTA); the pH-dependent deprotonation at C-5’ with ensuing
chemistry that releases adenine and S-ribosylmethionine; and the pH-independent
racemization about the sulfomium to give the (R,S)-diastereomer (Figure IV.1).46
Decay studies of AdoMet and its selenium and tellurium analogues, Se-AdoMet and
Te-AdoMet, were carried out at 37 °C and constant ionic strength. The result showed
that Se-AdoMet degraded significantly in both pH-dependent processes, while
Te-AdoMet was inert to both pH-dependent processes at 37 °C and pH values ranging
from 0.5 to 12.4‘5

Based on the inertness of Te-AdoMet demonstrated in the decay studies, it was
assumed that Te-AdoMet cannot function as a cosubstarte for the activation of PF L by
PFL-AE as AdoMet does. If no turnover occurs, in the presence of Te-AdoMet,
various interactions can be probed in the ternary complex PFL-AE [4Fe-4S]'+/ PFL/
Te-AdoMet by ENDOR spectroscopy. Sample with labeled Te-AdoMet could be used
to detect the interaction between [4Fe-4S]1+ and Te-AdoMet in presence of the

substrate PFL. Sample with labeled PFL could probe the interaction between

31

[4F e-4S]1+ and the substrate PF L, which cannot be done with regular AdoMet due to
the turnover. Features of the ternary complex PFL-AE/PFL/Te-AdoMet, together with
information from complex PFL-AE/PFL(G734A)/AdoMet would provide

comprehensive insight into the activation process of PFL.

N“*2
+ o (’th
T42 0' + H3O N”
I I +
H 0 0H3 0H
Marina

()4
S-Rbosyhatlome Pa. 11 MTA

 

ll

 

 

 

Figure IV.1 Three degradation pathways of AdoMet

IV.2 Experimental methods

IV.2.1 Materials

Te-AdoMet was obtained as a gift from Dr. Squire Booker. It came as sulfate salt,
dissolved in water and stored at -80 °C. 5-Deazariboflavin was synthesized in our

laboratory. All other chemicals were obtained commercially and used as received.

IV.2.2 Activity assay of wild-type PF L with Te-AdoMet

Activity assay was performed as described in Chapter 11, section 11.2.6. Two

32

samples were prepared and test simultaneously: one with Te-AdoMet, the other with
AdoMet for control. To assay PFL’s activity, 695 uL of coupling mix and 5 uL of the

activated PF L, were combined in a quartz cuvette, the production of NADH was

monitor by UV-vis at 340 nm.

IV.2.3 EPR Spectroscopy of ternary complex PFL/PFL-AE/Te-AdoMet

EPR first—derivative spectra were obtained at X-band on a Bruker ESP300E
spectrometer equipped with a liquid He cryostat and a temperature controller from
Oxford Instruments. Spectra were recorded at 12 K for [3Fe-4S]1+ and [4Fe-4S]'+, and
at 60 K to detect glycyl radical. Spin concentrations in the protein samples were
determined by calibrating double integrals of the EPR spectra recorded under
nonsaturating conditions (i) with a standard sample of 0.1 mM Cu(11) and 1 mM
EDTA solution for the cluster signals, or (ii) with a 1.04 mM K2(SO3)2NO solution for
the glycyl radical signals.

To prepare an EPR sample of the ternary complex PF L/PFL-AE/T e-AdoMet, all
procedures were carried out anaerobically in an inert atmosphere glove box (Mbraun)
with 02 level 5 3 ppm. A11 buffers, solvents and bath water were deoxygenated using
a vacuum/inert gas manifold before being taken into the glove box. Solid chemicals
were pumped in as solids. Ice was pre-chilled with liquid nitrogen before pumping
into the glove box.

A PFL-AE photoreduction mix was prepared first in an eppendorf to a volume of

ZOOuL with the following: Tris (50 mM, pH 8.1), 5’-deazariboflavin (100 pM),

33

isolated PF L-AE (200 uM), DTT (1 mM), glycerol (20%, w/v). The sample was then
transferred to an EPR tube. The EPR tube was capped and inserted in a beaker tightly
packed with ice-water. The sample was illuminated on ice using a 500 W halogen
lamp for 30 min with the lamp situated about 5 cm from the beaker. Afier illumination,
two equivalents of Te-AdoMet (21.4 mM in sulfate solution) were added. Lastly, 200
uL of inactive PF L, mix was added to the EPR tube with the following components:
PFL (200 IIM), Tris ( 1M, pH 8.1), 5’-deazariboflavin (100 pM), oxamate (20 mM),
DTT (1 mM), glycerol (20%, w/v). The tube was then brought out of the glove box,

and stored in liquid nitrogen for EPR.

IV.3 Results and Discussion
1V.3.l Activity assay of wild-type PFL with Te-AdoMet

Activity assay of wild type PFL with Te-AdoMet was performed in an analogous
manner as done for AdoMet. Unexpectedly, sample of Te-AdoMet showed PFL
activity as well as AdoMet. 45 min after PFL’s activation, units of PFLal in the cuvette:
for AdoMet, 0.36 units; for Te-AdoMet, 0.04 units. These values were calculated
according to Equation (1) in Chapter 11, section 11.2.6. Since the purpose of this
experiment is to test whether Te-AdoMet can act as a cosubstrate, not to quantify the
specific activity of PFL, readings were not continued until saturation. This results
indicates though Te-AdoMet was inert in the three major degradation ways, it is
readily reductively cleaved by PFL and PFL—AE. The following EPR spectra further

confirmed that Te-AdoMet can act as cosubstrate for PFL/PFL-AE.

34

IV.3.3 EPR of ternary complex PFL/PFL-AE/Te-AdoMet

EPR spectra of PFL/PFL-AE/Te-AdoMet were recorded at 12 K (Figure IV.2) and
60 K (Figure IV.3). If PFL-AE was fully photoreduced to [4Fe-4S]l+ and all being
consumed to activate PFL in the presence of Te-SAM, no EPR signal should be
detected at 12 K (since only EPR-silent [4Fe-4S]2+ would be present at that time).
Therefore, the observed signal (g = 2.01, 1.92, 1.88) in the EPR spectrum at 12 K is
probably due to incomplete reduction of PF L-AE. Compared to the nearly axial signal
(g = 2.01, 1.88, 1.87) for photoreduced PFL-AE in the presence of SAM,34 the
deviation of signal in Figure IV.2 may come from different binding interaction of
PF L-AE with Te-AdoMet. Or it may also be a result of PFL’s presence, since PFL is
expected to have close association with PFL-AB and AdoMet as well.

Spectrum at 60 K (Figure IV.3) exhibited a signal typical of glycyl radical (g =
2.00, a doublet splitting as seen in Figure 1V.4). Due to high noise and low
concentration, the signal was not sharp as usual, yet it confirmed the activation of PFL

in the presence of Te-AdoMet.

35

l L L A l

EPR Intensitv

 

 

 

 

 

vvv‘v1vvr11rvvvtvvvwvw—rv '1V‘TIVY'T'YVV'I'VVY'V'VV'f‘YVIVIVI'V'VV‘VVV‘V'YYYV

1000 1400 1800 2200 2600 3000 3400 3800
Field (Gauss)

Figure IV.2 X-band EPR spectrum of ternary complex

PFL/PFL-AE/Te-AdoMet. g values: 2.01, 1.92, 1.88. Conditions of

measurement, T=12K; microwave fi'equency, 9.47 GHz; modulation

amplitude, 10.084 G; modulation frequency, 100 KHz, 3 scans.

 

 

3 l
“a .
g l
E .
n:
n.
m .
l
1000 1400 1800 2200 2600 3000 3400 3800
Field (Gauss)

Figure IV.3 X-band EPR spectrum of ternary complex
PFL/PFL-AE/Te-AdoMet. g values:2.00. Conditions of measurement,

=60K; microwave frequency, 9.47 GHz; modulation amplitude, 10.084
G; modulation frequency, 100 KHz, 3 scans.

36

EPR Intensitv

 

 

3200 3240 3280 3320 3360 3400 3440 3480
Field (Gauss)

Figure IV.4 Blow-up of Figure IV.3 at the signal site. Details of the
doublet splitting can be seen from field 3200 Gauss to 3500 Gauss.

37

CHAPTER V

CONCLUSIONS

To probe one central question in the activation of pyruvate fomlate-lyase--how
PF L-AE recognizes glycine 734, the active site of PFL, one mutant PFL(G734A) in
plasmid pKK has been successfully overexpressed from E. coli. The enzyme was
purified by three chromatography methods in the order of ion exchange, gel filtration
and hydrophobic interaction. Purified mutant was used to perform the activity assay
of PFL. Unexpectedly, activity of 0.685 unit/mg was observed, which is
approximately 0.5% of the wild-type value. Since mutant PF L(G734A) is presumably
inactive, the weak activity observed is very likely due to trace amount of wild-type
PFL remained, which is difficult to be removed via normal purification methods.

In order to obtain more pure PFL(G734A), several different attempts have been
made. One is to clone PFL(G734A) gene into plasmids with an N-tenninal His-tag,
subsequently the mutant can be separated from wild-type via affinity chromatography.
Another method is the usage of knockout strain of E. coli, in which wild-type PF L
gene had already been deleted, thus transformation of mutant gene PFL(G734A) into
knockout strain will express only the mutant PFL(G734A). Difficulties were
encountered in the first method. PFL(G734A) with His-tag attached cannot be
purified successfully. 1n the second method, further effort needs to be taken to make
the cells chemically competent.

One analogue to the cosubstrate AdoMet, Te-AdoMet, was presumably inert for
the activation of PFL. However, both activity assays of Te-AdoMet and EPR spectra
of ternary complex PFL-AE/Te-AdoMet/PFL demonstrated that Te-AdoMet can

function as a cosubstrate for the activation of PFL by PFL-AE.

38

Future work is directed to the preparation of PFL(G734A) from the knockout
strain of E. coli. The purified mutant will be used to perform activity assay, EPR,
ENDOR of the ternary complex to probe the interaction among PFL-AE, AdoMet and

PF L, ultimately elucidate the radical generation process.

 

39

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42

 

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