This dissertation consists of three projects that dissect the nature of urease maturation and metal ion selectivity. The first project examines the role of the UreD accessory protein during in vivo maturation of the nickel-containing urease from Klebsiella aerogenes. A translational fusion of the maltose binding protein with UreD (MBP-UreD) was generated and found to be soluble. The UreD domain of MBP-UreD bound nickel and zinc ions, formed complexes with (UreABC)3, UreF, UreG, UreF plus UreG, and (UreABC)3-UreF-UreG in vivo, and formed a complex with the UreF domain of the UreE-UreF fusion in vitro. MBP-UreD was shown to be a functional form of UreD as malE-ureD partially complemented for a ∆ureD urease cluster. The second project revealed several roles for the K. aerogenes urease structural subunit UreB during urease maturation. UreB was purified as a monomer and shown to spontaneously bind to isolated (UreAC)3, forming (UreABC*)3, while an N-terminal deletion mutant of UreB lacking the first 19 residues did not form the apoprotein complex. (UreABC*)3 shared similar in vitro activation properties as urease apoprotein preformed in vivo, whereas exposure of a mixture of (UreAC)3 and UreB∆1-19 to activation conditions led to negligible levels of active enzyme. Activity assays and metal analyses of various in vitro activated species demonstrated that UreB facilitates efficient incorporation of Ni2+ into the active site and protects the metal from chelators. Additional studies revealed that UreB interfaced with accessory proteins, and the N-terminus was critical for this process. Finally, UreB enhanced the stability of UreC against proteolytic cleavage by trypsin. The third project characterized a unique urease from Helicobacter mustelae, UreA2B2, which was shown to exhibit O2-labile activity in whole cells. UreA2B2 was purified aerobically from its native host and found to contain ~ 2 iron per active site, or ~1 iron and 0.7 zinc when purified under anaerobic conditions. Anaerobically purified UreA2B2 was active, though highly O2-labile, with its activity enhanced by EDTA and inhibited by acetohydroxamic acid or nickel ions. The inactive, oxidized enzyme was slowly reactivated by incubation with dithionite to levels approaching the wild-type enzyme accompanied by bleaching of its UV-visible spectrum, where the chromophore was consistent with μ-oxo bridged diferric atoms. Resonance Raman spectroscopy of this sample revealed bands at ~500 cm-1 and ~780 cm-1 that are characteristic of a Fe(III)-O-Fe(III) metallocenter. The ~500 cm-1 feature was sensitive to bulk solvent exchange with H218O or deuterium oxide, and both features were downshifted in the presence of urea. Protein purified aerobically from recombinant Escherichia coli grown in rich medium contained ~1 equivalent of iron and negligible levels of other metals, while E. coli cultured in minimal medium generated apoprotein with ~0.2 equivalents of iron and 0.0-0.2 zinc. Temperature-dependent circular dichroism measurements indicated that iron enhanced the thermal stability of UreA2B2. The apoprotein form of the enzyme was activated to levels representing ~20% of wild-type activity with ferrous ions and bicarbonate under anaerobic conditions. Lastly, the crystal structure of UreA2B2 was determined at 3.0 Å revealing an active site architecture nearly identical to that for nickel ureases. Numerous amino acid residue substitutions around the active site suggest that metal specificity for iron likely arises during the metal loading process.
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