Biotin Sulfoxide Reductase Research Articles

NarJ subfamily system specific chaperone diversity and evolution is directed by respiratory enzyme associations.

BackgroundRedox enzyme maturation proteins (REMPs) describe a diverse family of prokaryotic chaperones involved in the biogenesis of anaerobic complex iron sulfur molybdoenzyme (CISM) respiratory systems. Many REMP family studies have focused on NarJ subfamily members from Escherichia coli: NarJ, NarW, DmsD, TorD and YcdY. The aim of this bioinformatics study was to expand upon the evolution, distribution and genetic association of these 5 REMP members within 130 genome sequenced taxonomically diverse species representing 324 Prokaryotic sequences. NarJ subfamily member diversity was examined at the phylum-species level and at the amino acid/nucleotide level to determine how close their genetic associations were between their respective CISM systems within phyla.ResultsThis study revealed that NarJ members possessed unique motifs that distinguished Gram-negative from Gram-positive/Archaeal species and identified a strict genetic association with its nitrate reductase complex (narGHI) operon compared to all other members. NarW appears to be found specifically in Gammaproteobacteria. DmsD also showed close associations with the dimethylsulfoxide reductase (dmsABC) operon compared to TorD. Phylogenetic analysis revealed that YcdY has recently evolved from DmsD and that YcdY has likely diverged into 2 subfamilies linked to Zn- dependent alkaline phosphatase (ycdX) operons and a newly identified operon containing part of Zn-metallopeptidase FtsH complex component (hflC) and NADH-quinone dehydrogenase (mdaB). TorD demonstrated the greatest diversity in operon association. TorD was identifed within operons from either trimethylamine-N-oxide reductase (torAC) or formate dehydrogenase (fdhGHI), where each type of TorD had a unique motif. Additionally a subgroup of dmsD and torD members were also linked to operons with biotin sulfoxide (bisC) and polysulfide reductase (nrfD) indicating a potential role in the maturation of diverse CISM.ConclusionExamination of diverse prokaryotic NarJ subfamily members demonstrates that the evolution and genetic association of each member is uniquely biased by its CISM operon association.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0412-3) contains supplementary material, which is available to authorized users.

Twin-Arginine Translocation System in Helicobacter pylori: TatC, but Not TatB, Is Essential for Viability

ABSTRACTThe twin-arginine translocation (Tat) system, needed to transport folded proteins across biological membranes, has not been characterized in the gastric pathogen Helicobacter pylori. Analysis of all H. pylori genome sequences available thus far reveals the presence of single copies of tatA, tatB, and tatC needed for the synthesis of a fully functional Tat system. Based on the presence of the twin-arginine hallmark in their signal sequence, only four H. pylori proteins appear to be Tat dependent: hydrogenase (HydA), catalase-associated protein (KapA), biotin sulfoxide reductase (BisC), and the ubiquinol cytochrome oxidoreductase Rieske protein (FbcF). In the present study, targeted mutations were aimed at tatA, tatB, tatC, or queA (downstream gene control). While double homologous recombination mutations in tatB and queA were easily obtained, attempts at disrupting tatA proved unsuccessful, while deletion of tatC led to partial mutants following single homologous recombination, with cells retaining a chromosomal copy of tatC. Double homologous recombination tatC mutants were obtained only when a plasmid-borne, isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible copy of tatC was introduced prior to transformation. These conditional tatC mutants could grow only in the presence of IPTG, suggesting that tatC is essential in H. pylori. tatB and tatC mutants had lower hydrogenase and catalase activities than the wild-type strain did, and the ability of tatC mutants to colonize mouse stomachs was severely affected compared to the wild type. Chromosomal complementation of tatC mutants restored hydrogenase and catalase activities to wild-type levels, and additional expression of tatC in wild-type cells resulted in elevated Tat-dependent enzyme activities. Unexpectedly, the tat strains had cell envelope defects.

Biotin sulfoxide reductase contributes to oxidative stress tolerance and virulence in Salmonella enterica serovar Typhimurium

Oxidative stress converts sulfur residues of molecules like biotin and methionine into their oxidized forms. Here we show that the biotin sulfoxide reductase BisC of Salmonella enterica serovar Typhimurium (S. Typhimurium) repairs both oxidized biotin and oxidized methionine. Exposure to H2O2 in vitro reduced survival of a S. Typhimurium ΔbisC mutant. Furthermore, replication of the ΔbisC mutant inside IFN-γ activated macrophages was reduced. In vitro tolerance of the mutant to H2O2 was restored by plasmids carrying either bisC or msrA; the latter encodes a methioinine sulfoxide reductase. In contrast, the proliferation defect inside IFN-γ activated macrophages was rescued by bisC but not by msrA. Thus growth of the ΔbisC mutant in IFN-γ activated macrophages required repair of oxidized biotin. Both the ΔbisC and a biotin auxotrophic (ΔbioB) mutant were attenuated in mice, suggesting that besides biotin biosynthesis, biotin repair was essential for virulence of S. Typhimurium in vivo. Attenuation of the ΔbisC mutant was more pronounced in 129 mice that produce a stronger oxidative response. These results show that BisC is essential for full virulence of Salmonella by contributing to the defence of S. Typhimurium against host-derived stress, and provides an attractive drug target since it is not present in mammals.

Remnant signal peptides on non-exported enzymes: implications for the evolution of prokaryotic respiratory chains

The twin-arginine translocation (Tat) pathway is a prokaryotic protein targeting system dedicated to the transmembrane translocation of folded proteins. Substrate proteins are directed to the Tat translocase by signal peptides bearing a conserved SRRxFLK 'twin-arginine' motif. In Escherichia coli, most of the 27 periplasmically located Tat substrates are cofactor-containing respiratory enzymes, and many of these harbour a molybdenum cofactor at their active site. Molybdenum cofactor-containing proteins are not exclusively located in the periplasm, however, with the major respiratory nitrate reductase (NarG) and the biotin sulfoxide reductase (BisC), for example, being located at the cytoplasmic side of the membrane. Interestingly, both NarG and BisC contain 'N-tail' regions that bear some sequence similarity to twin-arginine signal peptides. In this work, we have examined the relationship between the non-exported N-tails and the Tat system. Using a sensitive genetic screen for Tat transport, variant N-tails were identified that displayed Tat transport activity. For the NarG 36-residue N-tail, six amino acid changes were needed to induce transport activity. However, these changes interfered with binding by the NarJ biosynthetic chaperone and impaired biosynthesis of the native enzyme. For the BisC 36-residue N-tail, only five amino acid substitutions were needed to restore Tat transport activity. These modifications also impaired in vivo BisC activity, but it was not possible to identify a biosynthetic chaperone for this enzyme. These data highlight an intimate genetic and evolutionary link between some non-exported redox enzymes and those transported across membranes by the Tat translocation system.

YcbX and yiiM, two novel determinants for resistance of Escherichia coli to N-hydroxylated base analogues.

We have shown previously that lack of molybdenum cofactor (MoCo) in Escherichia coli leads to hypersensitivity to the mutagenic and toxic effects of N-hydroxylated base analogues, such as 6-N-hydroxylaminopurine (HAP). However, the nature of the MoCo-dependent mechanism is unknown, as inactivation of all known and putative E. coli molybdoenzymes does not produce any sensitivity. Presently, we report on the isolation and characterization of two novel HAP-hypersensitive mutants carrying defects in the ycbX or yiiM open reading frames. Genetic analysis suggests that the two genes operate within the MoCo-dependent pathway. In the absence of the ycbX- and yiiM-dependent pathways, biotin sulfoxide reductase plays also a role in the detoxification pathway. YcbX and YiiM are hypothetical members of the MOSC protein superfamily, which contain the C-terminal domain (MOSC) of the eukaryotic MoCo sulphurases. However, deletion of ycbX or yiiM did not affect the activity of human xanthine dehydrogenase expressed in E. coli, suggesting that the role of YcbX and YiiM proteins is not related to MoCo sulphuration. Instead, YcbX and YiiM may represent novel MoCo-dependent enzymatic activities. We also demonstrate that the MoCo/YcbX/YiiM-dependent detoxification of HAP proceeds by reduction to adenine.

Methionine Sulfoxide Reduction and Assimilation in Escherichia coli : New Role for the Biotin Sulfoxide Reductase BisC

Methionine ranks among the amino acids most sensitive to oxidation, which converts it to a racemic mixture of methionine-S-sulfoxide (Met-S-SO) and methionine-R-sulfoxide (Met-R-SO). The methionine sulfoxide reductases MsrA and MsrB reduce free and protein-bound MetSO, MsrA being specific for Met-S-SO and MsrB for Met-R-SO. In the present study, we report that an Escherichia coli metB1 auxotroph lacking both msrA and msrB is still able to use either of the two MetSO enantiomers. This indicates that additional methionine sulfoxide reductase activities occur in E. coli. BisC, a poorly characterized biotin sulfoxide reductase, was identified as one of these new methionine sulfoxide reductases. BisC was purified and found to exhibit reductase activity with free Met-S-SO but not with free Met-R-SO as a substrate. Moreover, a metB1 msrA msrB bisC strain of E. coli was unable to use Met-S-SO for growth, but it retained the ability to use Met-R-SO. Mass spectrometric analyses indicated that BisC is unable to reduce protein-bound Met-S-SO. Hence, this study shows that BisC has an essential role in assimilation of oxidized methionines. Moreover, this work provides the first example of an enzyme that reduces free MetSO while having no activity on peptide-bound MetSO residues.

An Active Site Tyrosine Influences the Ability of the Dimethyl Sulfoxide Reductase Family of Molybdopterin Enzymes to Reduce S-Oxides

Dimethyl sulfoxide reductase (DMSOR), trimethylamine-N-oxide reductase (TMAOR), and biotin sulfoxide reductase (BSOR) are members of a class of bacterial oxotransferases that contain the bis(molybdopterin guanine dinucleotide)molybdenum cofactor. The presence of a Tyr residue in the active site of DMSOR and BSOR that is missing in TMAOR has been implicated in the inability of TMAOR, unlike DMSOR and BSOR, to utilize S-oxides. To test this hypothesis, Escherichia coli TMAOR was cloned and expressed at high levels, and site-directed mutagenesis was utilized to generate the Tyr-114 --> Ala and Phe variants of Rhodobacter sphaeroides DMSOR and insert a Tyr residue into the equivalent position in TMAOR. Although all of the mutants turn over in a manner similar to their respective wild-type enzymes, mutation of Tyr-114 in DMSOR results in a decreased specificity for S-oxides and an increased specificity for trimethylamine-N-oxide (Me(3)NO), with a greater change observed for DMSOR-Y114A. Insertion of a Tyr into TMAOR results in a decreased preference for Me(3)NO relative to dimethyl sulfoxide. Kinetic analysis and UV-visible absorption spectra indicate that the ability of DMSOR to be reduced by dimethyl sulfide is lost upon mutation of Tyr-114 and that TMAOR does not exhibit this activity even in the Tyr insertion mutant.

Kinetic and mechanistic properties of biotin sulfoxide reductase.

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase catalyzes the reduction of d-biotin d-sulfoxide (BSO) to biotin. Initial rate studies of the homogeneous recombinant enzyme, expressed in Escherichia coli, have demonstrated that the purified protein utilizes NADPH as a facile electron donor in the absence of any additional auxiliary proteins. We have previously shown [Pollock, V. V., and Barber, M. J. (1997) J. Biol. Chem. 272, 3355-3362] that, at pH 8 and in the presence of saturating concentrations of BSO, the enzyme exhibits, a marked preference for NADPH (k(cat,app) = 500 s(-1), K(m,app) = 269 microM, and k(cat,app)/K(m,app) = 1.86 x 10(6) M(-1) s(-1)) compared to NADH (k(cat,app) = 47 s(-1), K(m,app) = 394 microM, and k(cat,app)/K(m,app) = 1.19 x 10(5) M(-1) s(-1)). Production of biotin using NADPH as the electron donor was confirmed by both the disk biological assay and by reversed-phase HPLC analysis of the reaction products. The purified enzyme also utilized ferricyanide as an artificial electron acceptor, which effectively suppressed biotin sulfoxide reduction and biotin formation. Analysis of the enzyme isolated from tungsten-grown cells yielded decreased reduced methyl viologen:BSO reductase, NADPH:BSO reductase, and NADPH:FR activities, confirming that Mo is required for all activities. Kinetic analyses of substrate inhibition profiles revealed that the enzyme followed a Ping Pong Bi-Bi mechanism with both NADPH and BSO exhibiting double competitive substrate inhibition. Replots of the 1/v-axes intercepts of the parallel asymptotes obtained at several low concentrations of fixed substrate yielded a K(m) for BSO of 714 and 65 microM for NADPH. In contrast, utilizing NADH as an electron donor, the replots yielded a K(m) for BSO of 132 microM and 1.25 mM for NADH. Slope replots of data obtained at high concentrations of BSO yielded a K(i) for BSO of 6.10 mM and 900 microM for NADPH. Kinetic isotope studies utilizing stereospecifically deuterated NADPD indicated that BSO reductase uses specifically the 4R-hydrogen of the nicotinamide ring. Cyanide inhibited NADPH:BSO and NADPH:FR activities in a reversible manner while diethylpyrocarbonate treatment resulted in complete irreversible inactivation of the enzyme concomitant with molybdenum cofactor release, indicating that histidine residues are involved in cofactor-binding.

Serine 121 Is an Essential Amino Acid for Biotin Sulfoxide Reductase Functionality

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase (BSOR) catalyzes the reduction of d-biotin d-sulfoxide (BSO) to biotin, an important step in oxidized vitamin salvaging. In addition to BSO, the enzyme also catalyzes the reduction of a variety of other substrates, including methionine sulfoxide, with decreased efficiencies, suggesting a potential role as a general cell protector against oxidative damage. Recombinant BSOR, expressed as a glutathione S-transferase fusion protein, contains the molybdopterin guanine dinucleotide cofactor (MGD) as its sole prosthetic group, which is required for the reduction of BSO by either NADPH or reduced methyl viologen. Comparison of the amino acid sequences of BSOR and the closely related MGD-containing enzyme, dimethyl sulfoxide reductase, has indicated a number of conserved residues, including an active site serine residue, serine 121, which has been potentially identified as the fifth coordinating ligand of Mo in BSOR. Site-directed mutagenesis has been used to replace serine 121 with cysteine, threonine, or alanine residues in the BSOR sequence to asses the role of this residue in catalysis and/or Mo coordination. All three BSOR mutant proteins were expressed, purified to homogeneity, and demonstrated to contain both MGD by fluorescence spectroscopy and Mo by inductively coupled plasma mass spectrometry, similar to wild-type enzyme. However, all three mutant proteins were devoid of BSOR activity using either NADPH or reduced methyl viologen as the electron donor. These results strongly suggest that serine 121 in BSOR is essential for catalysis but is not essential for either Mo coordination or MGD binding.

The torYZ ( yecK bisZ ) Operon Encodes a Third Respiratory Trimethylamine N -Oxide Reductase in Escherichia coli

The bisZ gene of Escherichia coli was previously described as encoding a minor biotin sulfoxide (BSO) reductase in addition to the main cytoplasmic BSO reductase, BisC. In this study, bisZ has been renamed torZ based on the findings that (i) the torZ gene product, TorZ, is able to reduce trimethylamine N-oxide (TMAO) more efficiently than BSO; (ii) although TorZ is more homologous to BisC than to the TMAO reductase TorA (63 and 42% identity, respectively), it is located mainly in the periplasm as is TorA; (iii) torZ belongs to the torYZ operon, and the first gene, torY (formerly yecK), encodes a pentahemic c-type cytochrome homologous to the TorC cytochrome of the TorCAD respiratory system. Furthermore, the torYZ operon encodes a third TMAO respiratory system, with catalytic properties that are clearly different from those of the TorCAD and the DmsABC systems. The torYZ and the torCAD operons may have diverged from a common ancestor, but, surprisingly, no torD homologue is found in the sequences around torYZ. Moreover, the torYZ operon is expressed at very low levels under the conditions tested, and, in contrast to torCAD, it is not induced by TMAO or dimethyl sulfoxide.

Structure of the molybdenum site of Rhodobacter sphaeroides biotin sulfoxide reductase.

Conditions for heterologous expression of Rhodobacter sphaeroides biotin sulfoxide reductase in Escherichia coli were modified, resulting in a significant improvement in the yield of recombinant enzyme and enabling structural studies of the molybdenum center. Quantitation of the guanine and the molybdenum as compared to that found in R. sphaeroides DMSO reductase demonstrated the presence of the bis(MGD)molybdenum cofactor. UV-visible absorption spectra were obtained for the oxidized, NADPH-reduced, and dithionite-reduced enzyme. EPR spectra were obtained for the Mo(V) state of the enzyme. X-ray absorption spectroscopy at the molybdenum K-edge has been used to probe the molybdenum coordination of the enzyme. The molybdenum site of the oxidized protein possesses a Mo(VI) mono-oxo site (Mo=O at 1.70 A) with additional coordination by approximately four thiolate ligands at 2.41 A and probably one oxygen or nitrogen at 1.95 A. The NADPH- and dithionite-reduced Mo(IV) forms of the enzyme are des-oxo molybdenum sites with approximately four thiolates at 2.33 A and two different Mo-O/N ligands at 2.19 and 1.94 A.

Resonance Raman Characterization of Biotin Sulfoxide Reductase: COMPARING OXOMOLYBDENUM ENZYMES IN THE Me2SO REDUCTASE FAMILY

Resonance Raman spectroscopy has been used to define active site structures for oxidized Mo(VI) and reduced Mo(IV) forms of recombinant Rhodobacter sphaeroides biotin sulfoxide reductase expressed in Escherichia coli. On the basis of (18)O/(16)O labeling studies involving water and the alternative substrate dimethyl sulfoxide and the close correspondence to the resonance Raman spectra previously reported for dimethyl sulfoxide reductase (Garton, S. D., Hilton, J., Oku, H., Crouse, B. R., Rajagopalan, K. V., and Johnson, M. K. (1997) J. Am. Chem. Soc. 119, 12906-12916), vibrational modes associated with a terminal oxo ligand and the two molybdopterin dithiolene ligands have been assigned. The results indicate that the enzyme cycles between mono-oxo-Mo(VI) and des-oxo-Mo(IV) forms with both molybdopterin dithiolene ligands remaining coordinated in both redox states. Direct evidence for an oxygen atom transfer mechanism is provided by (18)O/(16)O labeling studies, which show that the terminal oxo group at the molybdenum center is exchangeable with water during redox cycling and originates from the substrate in substrate-oxidized samples. Biotin sulfoxide reductase is not reduced by biotin or the nonphysiological products, dimethyl sulfide and trimethylamine. However, product-induced changes in the Mo=O stretching frequency provide direct evidence for a product-associated mono-oxo-Mo(VI) catalytic intermediate. The results indicate that biotin sulfoxide reductase is thermodynamically tuned to catalyze the reductase reaction, and a detailed catalytic mechanism is proposed.

Biotin Sulfoxide Reductase: HETEROLOGOUS EXPRESSION AND CHARACTERIZATION OF A FUNCTIONAL MOLYBDOPTERIN GUANINE DINUCLEOTIDE-CONTAINING ENZYME

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase has been heterologously expressed in Escherichia coli as a functional 106-kDa glutathione S-transferase fusion protein. Following cleavage with Factor Xa and purification to homogeneity, the soluble 83-kDa enzyme retained biotin sulfoxide reductase activity using reduced methyl viologen or reduced benzyl viologen as artificial electron donors. Initial rate kinetics indicated a specific activity at pH 8.0 of 0.9 micromol of biotin sulfoxide reduced per min/nmol of enzyme and Km values of 29 and 15 microM for reduced methyl viologen and biotin sulfoxide reductase, respectively. Biotin sulfoxide reductase was also capable of reducing nicotinamide N-oxide, methionine sulfoxide, trimethylamine-N-oxide, and dimethyl sulfoxide, although with varying efficiencies, and could directly utilize NADPH as a reducing agent, both for the reduction of biotin sulfoxide and ferricyanide. The enzyme contained the prosthetic group, molybdopterin guanine dinucleotide, and did not require any accessory proteins for functionality. These results represent the first successful heterologous expression and characterization of a functional molybdopterin guanine dinucleotide-containing enzyme and the demonstration of reduced pyridine nucleotide-dependent biotin sulfoxide reductase activity.

Alternative gene for biotin sulfoxide reduction in Escherichia coli K-12.

A gene at 42 min on the Escherichia coli chromosome, identified as the locus of pseudoreversion of knockout mutations in the biotin sulfoxide reductase gene, bisC, has 64% base sequence identity with bisC. This makes it a member of a multigene family of molybdopterin enzymes that includes genes for anaerobic reduction of trimethylamine oxide (torA) and dimethylsulfoxide (dmsA). Disruption of this gene eliminates the background activity of biotin sulfoxide reduction observed in bisC mutants. Sequence comparison of the new gene (bisZ) with bisC indicates that certain ts mutants of bisC arise by gene conversion between the two loci.

Molecular Cloning and Expression of Biotin Sulfoxide Reductase from Rhodobacter sphaeroides Forma Sp. Denitrificans

Biotin sulfoxide reductase catalyzes the conversion of d-biotin d-sulfoxide (BSO) to d-biotin. Oligonucleotides directed against common sequences in Escherichia coli biotin sulfoxide reductase and in Rhodobacter sphaereides f.sp. denitrificans dimethyl sulfoxide reductase have been utilized to amplify by PCR a 651-bp fragment from R. sphaeroides total genomic DNA that showed a high degree of sequence similarity with both E. coli biotin sulfoxide reductase and R. sphaeroides dimethyl sulfoxide reductase. Screening of a genomic cosmid library, prepared from R. sphaeroides genomic DNA, with this probe resulted in the isolation of a 7-kb EcoRI- EcoRI fragment that contained the complete coding region for R. sphaereides BSO reductase which has been sequenced, The sequence data indicated a single open reading frame of 2231 nucleotides encoding a protein of 744 amino acid residues corresponding to a subunit molecular weight of 80,234 Da. The translated protein sequence contained the prokaryotic Mo-pterin signatures 2 and 3 (Mo-cofactor binding motifs) and a ATP/GTP-binding P-loop, The R. sphaeroides BSO reductase sequence showed 51% sequence similarity with the corresponding E. coli enzyme. In addition, there were only two conserved cysteines between the two BSO reductase sequences. The R. sphaeroides gene was demonstrated, by complementation, to rescue a mutant E. coli strain that was deficient in BSO reductase when grown on BSO as the sole source of biotin, When expressed from the FLAG*Shift 12c expression vector, in the presence of IPTG, the BSO reductase gene encoded a protein of approximately 80 kDa, which cross-reacted with the anti-FLAG: monoclonal antibody and exhibited BSO reductase activity by the disk microbiological assay.

Cloning and nucleotide sequence of the gene encoding dimethyl sulfoxide reductase from Rhodobacter sphaeroides f. sp. denitrificans.

The gene encoding dimethyl sulfoxide (DMSO) reductase, which contains a molybdenum cofactor, of the phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans was isolated using an oligonucleotide probe, which was synthesized based on a internal amino acid sequence of the purified enzyme. The DMSO reductase gene coded for 822 amino acids (2466 base pairs, M(r) = 89,206) as a precursor form having a signal peptide of 42 amino acids. The deduced amino acid sequence had high homology with those of some enzymes containing a molybdenum cofactor: trimethyl amine N-oxide reductase (48%), biotin sulfoxide reductase (44%), and DMSO reductase (29%) of Escherichia coli.

Cloning and nucleotide sequence of bisC, the structural gene for biotin sulfoxide reductase in Escherichia coli

Clones of the Escherichia coli bisC locus have been isolated by complementing a bisC mutant for growth with d-biotin d-sulfoxide as a biotin source. The complementation properties of deletions and Tn5 insertions located the bisC gene to a 3.7-kilobase-pair (kbp) segment, 3.3 kbp of which has been sequenced. A single open reading frame of 2,178 bp, capable of encoding a polypeptide of molecular weight 80,905, was found. In vitro transcription of plasmids carrying the wild-type sequence and deletion and insertion mutants showed that BisC complementation correlated perfectly with production of a polypeptide whose measured molecular weight (79,000) does not differ significantly from 80,905.

Molybdate reduction by Escherichia coli K-12 and its chl mutants.

During anaerobic growth, Escherichia coli can reduce phosphomolybdate. The reduction can also be carried out by washed cells suspended in buffer at pH 5.7. Phosphate, molybdate, glucose, cells, and anaerobic conditions are required. Reduction is inhibited by 200 microM chromate, 290 microM nitrite, 10 mM tungstate, or 20 mM cysteine. Wild-type (chl+) cells are inhibited by addition of 200 microM nitrate, but chlA, chlB, and chlE mutants are not. The inhibition of chl+ cells results from reduction of nitrate to nitrite. This nitrate reduction is not catalyzed by nitrate reductase. Wild-type cells are more sensitive than chl mutants to inhibition by nitrite and cysteine but more resistant to chromate. Pregrowth of chlD cells in 1 mM Na2MoO4 increases their sensitivity to nitrite and cysteine, and pregrowth of chl+ cells in 1 mM Na2MoO4 increases their resistance to these agents. Assays of biotin sulfoxide reductase show that the tightness of the chlD block depends on growth conditions; chlD cells grown aerobically in tryptone broth make about 50% as much active enzyme as chl+ cells, whereas chlD cells grown anaerobically with tryptone plus glucose make less than 10%. The effect of anaerobic pregrowth on the inhibition of molybdate reduction by added nitrate indicates that in vivo nitrate reduction responds to growth conditions in the same manner as biotin sulfoxide reductase does.

Molybdenum cofactor requirement for biotin sulfoxide reduction in Escherichia coli.

The bisC gene of Escherichia coli is tentatively identified as the structural gene for biotin sulfoxide reductase by the isolation of bisC(Ts) mutants that make thermolabile enzyme. The products of four other E. coli genes (chlA, chlB, chlE and chlG) are also needed for enzymatic activity. Mutations previously assigned to the bisA, bisB, and bisD genes belong to genes chlA, chlE, and chlG, respectively. The biotin sulfoxide reductase deficiency of a chlG, mutant is partially reversed by the addition of 10 mM molybdate to the growth medium. Mutational inactivation of the chlD gene reduces the specific activity of biotin sulfoxide reductase about twofold. This effect is reversed by the addition of 1 mM molybdate to the growth medium. The specific activity of biotin sulfoxide reductase is decreased about 30-fold by the presence of tungstate in the growth medium, an effect that has been observed previously with nitrate reductase and other molybdoenzymes. The specific activity of biotin sulfoxide reductase is not elevated in a lysate prepared by derepressing a lambda cI857 chlG prophage. Whereas biotin sulfoxide reductase prepared by sonic extraction of growing cells is almost completely dependent on the presence of a small heat-stable protein resembling thioredoxin, much of the enzyme obtained from lysates of thermoinduced lambda cI857 lysogens does not require this factor.