The identification of a novel protein involved in molybdenum cofactor biosynthesis in Escherichia coli.

In the second step of the molybdenum cofactor (Moco) biosynthesis in Escherichia coli, the l-cysteine desulfurase IscS was identified as the primary sulfur donor for the formation of the thiocarboxylate on the small subunit (MoaD) of MPT synthase, which catalyzes the conversion of cyclic pyranopterin monophosphate to molybdopterin (MPT). Although in Moco biosynthesis in humans, the thiocarboxylation of the corresponding MoaD homolog involves two sulfurtransferases, an l-cysteine desulfurase, and a rhodanese-like protein, the rhodanese-like protein in E. coli remained enigmatic so far. Using a reverse approach, we identified a so far unknown sulfurtransferase for the MoeB-MoaD complex by protein-protein interactions. We show that YnjE, a three-domain rhodanese-like protein from E. coli, interacts with MoeB possibly for sulfur transfer to MoaD. The E. coli IscS protein was shown to specifically interact with YnjE for the formation of the persulfide group on YnjE. In a defined in vitro system consisting of MPT synthase, MoeB, Mg-ATP, IscS, and l-cysteine, YnjE was shown to enhance the rate of the conversion of added cyclic pyranopterin monophosphate to MPT. However, YnjE was not an enhancer of the cysteine desulfurase activity of IscS. This is the first report identifying the rhodanese-like protein YnjE as being involved in Moco biosynthesis in E. coli. We believe that the role of YnjE is to make the sulfur transfer from IscS for Moco biosynthesis more specific because IscS is involved in a variety of different sulfur transfer reactions in the cell.

Enterococcus faecalis sufCDSUB complements Escherichia coli sufABCDSE.

Iron-sulfur [Fe-S] clusters are inorganic prosthetic groups that play essential roles in all living organisms. Iron and sulfur mobilization, formation of [Fe-S] clusters, and delivery to its final protein targets involves a complex set of specific protein machinery. Proteobacteria has three systems of [Fe-S] biogenesis, designated NIF, ISC, and SUF. In contrast, the Firmicutes system is not well characterized and has only one system, formed mostly by SUF homologs. The Firmicutes phylum corresponds to a group of pathological bacteria, of which Enterococcus faecalis is a clinically relevant representative. Recently, the E. faecalis sufCDSUB [Fe-S] cluster biosynthetic machinery has been identified, although there is no further information available about the similarities and/or variations of Proteobacteria and Firmicutes systems. The aim of the present work was to compare the ability of the different Proteobacteria and Firmicutes systems to complement the Azotobacter vinelandii and Escherichia coli ISC and SUF systems. Indeed, E. faecalis sufCDSUB is able to complement the E. coli SUF system, allowing viable mutants of both sufABCDSE and iscRSU-hscBA-fdx systems. The presence of all E. faecalis SUF factors enables proper functional interactions, which would not otherwise occur in proteins from different systems.

Genome sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes.

Azotobacter vinelandii is a soil bacterium related to the Pseudomonas genus that fixes nitrogen under aerobic conditions while simultaneously protecting nitrogenase from oxygen damage. In response to carbon availability, this organism undergoes a simple differentiation process to form cysts that are resistant to drought and other physical and chemical agents. Here we report the complete genome sequence of A. vinelandii DJ, which has a single circular genome of 5,365,318 bp. In order to reconcile an obligate aerobic lifestyle with exquisitely oxygen-sensitive processes, A. vinelandii is specialized in terms of its complement of respiratory proteins. It is able to produce alginate, a polymer that further protects the organism from excess exogenous oxygen, and it has multiple duplications of alginate modification genes, which may alter alginate composition in response to oxygen availability. The genome analysis identified the chromosomal locations of the genes coding for the three known oxygen-sensitive nitrogenases, as well as genes coding for other oxygen-sensitive enzymes, such as carbon monoxide dehydrogenase and formate dehydrogenase. These findings offer new prospects for the wider application of A. vinelandii as a host for the production and characterization of oxygen-sensitive proteins.

Biochemical and Genetic Characterization of PspE and GlpE, Two Single-domain Sulfurtransferases of Escherichia coli.

The pspE and glpE genes of Escherichia coli encode periplasmic and cytoplasmic single-domain rhodaneses, respectively, that catalyzes sulfur transfer from thiosulfate to thiophilic acceptors. Strains deficient in either or both genes were constructed. Comparison of rhodanese activity in these strains revealed that PspE provides 85% of total rhodanese activity, with GlpE contributing most of the remainder. PspE activity was four times higher during growth on glycerol versus glucose, and was not induced by conditions that induce expression of the psp regulon. The glpE/pspE mutants displayed no apparent growth phenotypes, indicating that neither gene is required for biosynthesis of essential sulfur-containing molecules. PspE was purified by using cation exchange chromatography. Two distinct active peaks were eluted and differed in the degree of stable covalent modification, as assessed by mass spectrometry. The peak eluting earliest contained the equivalent mass of two additional sulfur atoms, whereas the second peak contained mainly one additional sulfur. Kinetic properties of purified PspE were consistent with catalysis occurring via a double-displacement mechanism via an enzyme-sulfur intermediate involving the active site cysteine. K(m)s for SSO(3) (2-) and CN(-) were 2.7 mM and 32 mM, respectively, and k(cat) was 64(s-1). The enzyme also catalyzed transfer of sulfur from thiosulfate to dithiothreitol, ultimately releasing sulfide.

Correlation between protein binding strength on immobilized metal affinity chromatography and the histidine-related protein surface structure.

Immobilized metal affinity chromatography (IMAC) was investigated for its ability to characterize the histidine-related surface structure of a protein, that is, a histidine residue's surface accessibility and its potential involvement in intramolecular interactions. T4 lysozyme was chosen as the model protein. Seven amino acid sites were selected on the basis of their relative surface accessibility, and they were substituted with histidine via site-directed protein mutagenesis to generate seven T4 lysozyme variants, each containing only one histidine residue on its surface, with various surface accessibility. IMAC was then used to experimentally quantify the interaction of each lysozyme variant with immobilized copper ions. A direct correlation was shown between the protein binding affinity and the surface accessibility of the histidine residue. Of all the lysozyme variants, K83H and K147H showed unusually low binding strength, as compared with variants having a histidine residue with a similar surface accessibility. However, with the aid of molecular modeling, their relatively low binding affinities were predicted to be the result of the involvement of the histidine residue in intramolecular interactions. In contrast to previously reported results, our results showed that lysozyme still binds to the IMAC column, even if its histidine residue is involved in intramolecular bonding, such as a hydrogen bond, albeit at reduced strength, as compared with the variant containing a histidine residue with a similar surface accessibility.

A phosphohexomutase from the archaeon Sulfolobus solfataricus is covalently modified by phosphorylation on serine.

A phosphoserine-containing peptide was identified from tryptic digests from Sulfolobus solfataricus P1 by liquid chromatography-tandem mass spectrometry. Its amino acid sequence closely matched that bracketing Ser-309 in the predicted protein product of open reading frame sso0207, a putative phosphohexomutase, in the genome of S. solfataricus P2. Open reading frame sso0207 was cloned, and its protein product expressed in Escherichia coli. The recombinant protein proved capable of interconverting mannose 1-phosphate and mannose 6-phosphate, as well as glucose 1-phosphate and glucose 6-phosphate, in vitro. It displayed no catalytic activity toward glucosamine 6-phosphate or N-acetylglucosamine 6-phosphate. Models constructed using the X-ray crystal structure of a homologous phosphohexomutase from Pseudomonas aeruginosa predicted that Ser-309 of the archaeal protein lies within the substrate binding site. The presence of a phosphoryl group at this location would be expected to electrostatically interfere with the binding of negatively charged phosphohexose substrates, thus attenuating the catalytic efficiency of the enzyme. Using site-directed mutagenesis, Ser-309 was substituted by aspartic acid to mimic the presence of a phosphoryl group. The V(max) of the mutationally altered protein was only 4% that of the unmodified form. Substitution of Ser-309 with larger, but uncharged, amino acids, including threonine, also decreased catalytic efficiency, but to a lesser extent--three- to fivefold. We therefore predict that phosphorylation of the enzyme in vivo serves to regulate its catalytic activity.

Application of AgaR repressor and dominant repressor variants for verification of a gene cluster involved in N-acetylgalactosamine metabolism in Escherichia coli K-12.

The agaZVWEFASYBCDI gene cluster encodes the phosphotransferase systems and enzymes responsible for the uptake and metabolism of N-acetylgalactosamine and galactosamine in Escherichia coli. In some strains of E. coli, particularly the common K-12 strain, a portion of this cluster is missing because of a site-specific recombination event that occurred between sites in agaW and agaA. Strains that have undergone this recombination event have lost the ability to utilize either N-acetylgalactosamine or galactosamine as sole sources of carbon. Divergently transcribed from this gene cluster is the gene agaR encoding a transcriptional repressor belonging to the DeoR/GlpR family of transcriptional regulators. Promoters upstream of agaR, agaZ and agaS were characterized. All three promoters had elevated activity in the presence of N-acetylgalactosamine or galactosamine, were regulated in vivo by AgaR and possessed specific DNA-binding sites for AgaR upstream from the start sites of transcription as determined by DNase I footprinting. In vivo analysis and DNase I footprinting indicated that the promoter specific for agaZ also requires activation by cAMP-CRP. Previous work with GlpR and other members of the DeoR/GlpR family have identified highly conserved amino acid residues that function in DNA-binding or response to inducer. These residues of AgaR were targeted for site-directed mutagenesis and yielded variants of AgaR that were either negatively dominant or non-inducible. The apparent ability to produce negatively dominant and non-inducible variants of proteins of the DeoR/GlpR family of currently unknown function will likely facilitate screening for function.

Functional diversity of the rhodanese homology domain: the Escherichia coli ybbB gene encodes a selenophosphate-dependent tRNA 2-selenouridine synthase.

Escherichia coli has eight genes predicted to encode sulfurtransferases having the active site consensus sequence Cys-Xaa-Xaa-Gly. One of these genes, ybbB, is frequently found within bacterial operons that contain selD, the selenophosphate synthetase gene, suggesting a role in selenium metabolism. We show that ybbB is required in vivo for the specific substitution of selenium for sulfur in 2-thiouridine residues in E. coli tRNA. This modified tRNA nucleoside, 5-methylaminomethyl-2-selenouridine (mnm(5)se(2)U), is located at the wobble position of the anticodons of tRNA(Lys), tRNA(Glu), and tRNA(1)(Gln). Nucleoside analysis of tRNAs from wild-type and ybbB mutant strains revealed that production of mnm(5)se(2)U is lost in the ybbB mutant but that 5-methylaminomethyl-2-thiouridine, the mnm(5)se(2)U precursor, is unaffected by deletion of ybbB. Thus, ybbB is not required for the initial sulfurtransferase reaction but rather encodes a 2-selenouridine synthase that replaces a sulfur atom in 2-thiouridine in tRNA with selenium. Purified 2-selenouridine synthase containing a C-terminal His(6) tag exhibited spectral properties consistent with tRNA bound to the enzyme. In vitro mnm(5)se(2)U synthesis is shown to be dependent on 2-selenouridine synthase, SePO(3), and tRNA. Finally, we demonstrate that the conserved Cys(97) (but not Cys(96)) in the rhodanese sequence motif Cys(96)-Cys(97)-Xaa-Xaa-Gly is required for 2-selenouridine synthase in vivo activity. These data are consistent with the ybbB gene encoding a tRNA 2-selenouridine synthase and identifies a new role for the rhodanese homology domain in enzymes.