Biochemical Studies on Mycobacterium tuberculosis UreG and Comparative Modeling Reveal Structural and Functional Conservation among the Bacterial UreG Family

Abstract

Nickel is a fundamental micronutrient for cellular life, but it is toxic in soluble form at nonphysiological concentrations. Such potentially contradictory features required living organisms to develop efficient systems for nickel utilization and homeostasis. This is the case for incorporation of nickel into the active site of urease, a multistep, tightly regulated process, requiring the interplay of various accessory proteins. The understanding of this activation mechanism may find medical applications against ureolytic bacteria, among which Mycobacterium tuberculosis is a deadly pathogen for humans. The topic of this study is UreG, an essential chaperone in the in vivo activation of urease upon insertion of Ni2+ into the active site. The protein was examined using both experimental and computational approaches. In particular, the soluble M. tuberculosis UreG (MtUreG) was overexpressed in Escherichia coli and purified to homogeneity. The identity of the isolated protein was established by mass spectrometry. On-line size-exclusion chromatography and light scattering indicated that MtUreG exists as a dimeric form in solution. Determination of the free thiol concentration revealed that a disulfide bond is present in the dimer. The isolated MtUreG shows low GTPase activity under native conditions, with a kcat of 0.01 min-1. Circular dichroism spectroscopy demonstrated the presence of a well-defined secondary structure (8% alpha-helices, 29% beta-strands) in MtUreG, whereas NMR spectroscopy indicated that this protein does not behave as a rigid three-dimensional fold and thus can be assigned to the class of intrinsically unstructured polypeptides. The molecular model of MtUreG in the fully folded and functional form was built using fold recognition algorithms. An extensive similarity search was performed to determine conservation patterns in all known bacterial UreG sequences. The generation of a multiple-sequence alignment and the related phylogenetic tree allowed us to recognize key residues and motifs that are likely important for protein function. A structural database containing the homology-built models of the most representative UreG proteins was created, confirming the structural analogies among the UreG family. A flexible region, likely to be important for protein function, is identified. The structural conservation among this class of GTPases is discussed on the basis of their function in the urease assembly process.

Functional and Phylogenetic Analysis of ureD in Shiga Toxin-Producing Escherichia coli

“…A combination phylogenetic analysis based on single protein sequences and derived model protein structures has been used by one group in an attempt to study the evolutionary conservation of key residues or protein structures for the UreE, UreF, and UreG proteins (34,44,56). The protein sequence of UreC, the structural subunit containing the active site of urease and the largest of the urease proteins, has been used for phylogenetic characterization of ammonia-oxidizing bacteria (23) and in a study including 22 diverse bacterial species for phylogenetic characterization of the two ure gene clusters found in Brucella suis (7).…”

Biosynthesis of the Urease Metallocenter

“…UreG: GTPase for Urease Activation-In contrast to UreD/ UreH and K. aerogenes UreF, which are insoluble, UreG is soluble and has been characterized from several sources (43)(44)(45)(46)(47) (46). X-ray absorption spectroscopy of the zincbound protein revealed a trigonal bipyramidal site including two His and two Cys residues, likely positioned at the subunit interface (49).…”

IcmF Is a Fusion between the Radical B12 Enzyme Isobutyryl-CoA Mutase and Its G-protein Chaperone

“…In comparison, for isobutyryl-CoA a K m of 57 Ϯ 13 M has been reported for ICM from S. cinnamonensis (2 . In B 12 proteins that bind the cofactor in a base-off/His-on conformation, a signature …SXL…GG motif is found (highlighted in blue). In IcmF, this motif is similar but not identical as follows: DXHXX(A/S)…(S/ T)XY…GGGG (highlighted in red).…”

“…B 12 (or 5Ј-deoxyadenosylcobalamin (AdoCbl))-dependent enzyme, which catalyzes the rearrangement of isobutyryl-CoA to n-butyryl-CoA (1)(2)(3). This reaction is very similar to that catalyzed by methylmalonyl-CoA mutase (MCM), which is better studied and more widely distributed in nature (4).…”

“…MeaB from Methylobacterium extorquens has been characterized most extensively and is a P-loop GTPase (9,10). Other members of this subfamily include HypB, UreG, and CooC, which are important in the assembly of the following metalloenzymes: nickel hydrogenases (11), urease (12), and CO dehydrogenase (13), respectively. MeaB has been proposed to function in the GTP-dependent assembly of holo-MCM and shown to protect the radical intermediates formed during MCM catalysis from oxidative interception (14).…”