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.     

Characterization of molybdenum cofactor from Escherichia coli.

Molybdenum cofactor activity was found in the soluble fraction of cell-free extracts of Escherichia coli grown aerobically in media supplemented with molybdate. Cofactor was detected by its ability to complement the nitrate reductase-deficient mutant of Neurospora crossa, nit-1, resulting in the vitro formation of nitrate reductase activity. Acid treatment of E. coli extracts was not required for release of cofactor activity. Cofactor was able to diffuse through a membrane of nominal 2,000-molecular-weight cutoff and was insensitive to trypsin. The cofactor was associated with a carrier molecule (approximately 40,000 daltons) during gel filtration and sucrose gradient centrifugation, but was easily removed from the carrier by dialysis. The carrier molecule protected the cofactor from inactivation by heat or oxygen. E. coli grown in molybdenum-free media, without and with tungsten, synthesized a metal-free "empty" cofactor and its tungsten analog, respectively, both of which were subsequently activated by the addition of molybdate. Empty and tungsten-containing cofactor complemented the nitrate reductase subunits in the nit-1 extract, forming inactive, but intact, 7.9S nitrate reductase. Addition of molybdate to the enzyme complemented in this manner restored nitrate reductase activity.     

Reductive cleavage of S-adenosylmethionine by biotin synthase from Escherichia coli

Biotin synthase (BioB) catalyzes the insertion of a sulfur atom between the C6 and C9 carbons of dethiobiotin. Reconstituted BioB from Escherichia coli contains a [4Fe-4S](2+/1+) cluster thought to be involved in the reduction and cleavage of S-adenosylmethionine (AdoMet), generating methionine and the reactive 5'-deoxyadenosyl radical responsible for dethiobiotin H-abstraction. Using EPR and M?ssbauer spectroscopy as well as methionine quantitation we demonstrate that the reduced S = 1/2 [4Fe-4S](1+) cluster is indeed capable of injecting one electron into AdoMet, generating one equivalent of both methionine and S = 0 [4Fe-4S](2+) cluster. Dethiobiotin is not required for the reaction. Using site-directed mutagenesis we show also that, among the eight cysteines of BioB, only three (Cys-53, Cys-57, Cys-60) are essential for AdoMet reductive cleavage, suggesting that these cysteines are involved in chelation of the [4Fe-4S](2+/1+) cluster.     

The PLP-dependent biotin synthase from Escherichia coli: mechanistic studies

Biotin synthase (BioB), an iron-sulfur enzyme, catalyzes the last step of the biotin biosynthesis pathway. The reaction consists in the introduction of a sulfur atom into two non-activated C-H bonds of dethiobiotin. Substrate radical activation is initiated by the reductive cleavage of S-adenosylmethionine (AdoMet) into a 5'-deoxyadenosyl radical. The recently described pyridoxal 5'-phosphate-bound enzyme was used to show that only one molecule of AdoMet, and not two, is required for the formation of one molecule of biotin. Furthermore 5'-deoxyadenosine, a product of the reaction, strongly inhibited biotin formation, an observation that may explain why BioB is not able to make more than one turnover. However this enzyme inactivation is not irreversible.     

Identification of the [Fe-S] cluster-binding residues of Escherichia coli biotin synthase

The gene encoding Escherichia coli biotin synthase (bioB) has been expressed as a histidine fusion protein, and the protein was purified in a single step using immobilized metal affinity chromatography. The His(6)-tagged protein was fully functional in in vitro and in vivo biotin production assays. Analysis of all the published bioB sequences identified a number of conserved residues. Single point mutations, to either serine or threonine, were carried out on the four conserved (Cys-53, Cys-57, Cys-60, and Cys-188) and one non-conserved (Cys-288) cysteine residues, and the purified mutant proteins were tested both for ability to reconstitute the [2Fe-2S] clusters of the native (oxidized) dimer and enzymatic activity. The C188S mutant was insoluble. The wild-type and four of the mutant proteins were characterized by UV-visible spectroscopy, metal and sulfide analysis, and both in vitro and in vivo biotin production assays. The molecular masses of all proteins were verified using electrospray mass spectrometry. The results indicate that the His(6) tag and the C288T mutation have no effect on the activity of biotin synthase when compared with the wild-type protein. The C53S, C57S, and C60S mutant proteins, both as prepared and reconstituted, were unable to covert dethiobiotin to biotin in vitro and in vivo. We conclude that three of the conserved cysteine residues (Cys-53, Cys-57, and Cys-60), all of which lie in the highly conserved "cysteine box" motif, are crucial for [Fe-S] cluster binding, whereas Cys-188 plays a hitherto unknown structural role in biotin synthase.     

Mutagenesis of the proposed iron-sulfur cluster binding ligands in Escherichia coli biotin synthase

Biotin synthase (BioB) is a member of a family of enzymes that includes anaerobic ribonucleotide reductase and pyruvate formate lyase activating enzyme. These enzymes all use S-adenosylmethionine during turnover and contain three highly conserved cysteine residues that may act as ligands to an iron-sulfur cluster required for activity. Three mutant enzymes of BioB have been made, each with one cysteine residue (C53, 57, 60) mutated to alanine. All three mutant enzymes were inactive, but they still exhibited the characteristic UV-visible spectrum of a [2Fe-2S]2+ cluster similar to that of the wild-type enzyme.     

Characterization of a specific interaction between Escherichia coli thymidylate synthase and Escherichia coli thymidylate synthase mRNA.

Previous studies have shown that human TS mRNA translation is controlled by a negative autoregulatory mechanism. In this study, an RNA electrophoretic gel mobility shift assay confirmed a direct interaction between Escherichia coli (E.coli) TS protein and its own E.coli TS mRNA. Two cis-acting sequences in the E.coli TS mRNA protein-coding region were identified, with one site corresponding to nucleotides 207-460 and the second site corresponding to nucleotides 461-807. Each of these mRNA sequences bind TS with a relative affinity similar to that of the full-length E.coli TS mRNA sequence (IC50 = 1 nM). A third binding site was identified, corresponding to nucleotides 808-1015, although its relative affinity for TS (IC50 = 5.1 nM) was lower than that of the other two cis-acting elements. E.coli TS proteins with mutations in amino acids located within the nucleotide-binding region retained the ability to bind RNA while proteins with mutations at either the nucleotide active site cysteine (C146S) or at amino acids located within the folate-binding region were unable to bind TS mRNA. These studies suggest that the regions on E.coli TS defined by the folate-binding site and/or critical cysteine sulfhydryl groups may represent important RNA binding domains. Further evidence is presented which demonstrates that the direct interaction with TS results in in vitro repression of E.coli TS mRNA translation.     

Characterization of Escherichia coli MoeB and its involvement in the activation of molybdopterin synthase for the biosynthesis of the molybdenum cofactor.

Amino acid sequence comparisons of Escherichia coli MoeB suggested that the MoeB-dependent formation of a C-terminal thiocarboxylate on the MoaD subunit of molybdopterin synthase might resemble the ubiquitin-activating step in the ubiquitin-targeted degradation of proteins in eukaryotes. To determine the exact role of MoeB in molybdopterin biosynthesis, the protein was purified after homologous overexpression. Using purified proteins, we have demonstrated the ATP-dependent formation of a complex of MoeB and MoaD adenylate that is stable to gel filtration. Mass spectrometry of the complex revealed a peak of a molecular mass of 9,073 Da, the expected mass of MoaD adenylate. However, unlike the ubiquitin activation reaction, the formation of a thioester intermediate between MoeB and MoaD could not be observed. There was also no evidence for a MoeB-bound sulfur during the sulfuration of MoaD. Amino acid substitutions were generated in every cysteine residue in MoeB. All of these exhibited activity comparable to the wild type, with the exception of mutations in cysteine residues located in putative Zn-binding motifs. For these cysteines, loss of activity correlated with loss of metal binding.     

Uptake of extracellular biotin by Escherichia coli biotin prototrophs.

Uptake of exogenous biotin by two Escherichia coli biotin prototroph strains, K-12 and Crookes, appeared to involve incorporation at a fixed number of binding sites located at the cell membrane. Incorporation was characterized as a binding process specific for biotin, not requiring energy, and stimulated by acidic pH. Constant saturation quantities of exogenous biotin were incorporated by these cells, and the amounts, which were titrated, depended on whether the cells were resting or dividing. Resting cells incorporated exogenous biotin amounting to 2% of their total intracellular biotin content. Fifty percent of the exogenous biotin was incorporated into their free biotin fraction, and 50% was incorporated into their bound biotin fraction. On the other hand, dividing cells incorporated exogenous biotin into all of their intracellular sites, 88% going into the intracellular-bound biotin fraction, and 12% going into the free biotin fraction. Calculations suggested that each cell contained approximately 3,000 binding sites for biotin. It was postulated that biotin incorporation sites might have been components of acetyl coenzyme A carboxylase located at or near the membrane.     

9-Mercaptodethiobiotin is formed as a competent catalytic intermediate by Escherichia coli biotin synthase

Biotin synthase (BS) catalyzes the oxidative addition of a sulfur atom to dethiobiotin (DTB) to generate the biotin thiophane ring. This enzyme is an S-adenosylmethionine (AdoMet) radical enzyme that catalyzes the reductive cleavage of AdoMet, generating methionine and a transient 5'-deoxyadenosyl radical. In our working mechanism, the 5'-deoxyadenosyl radical oxidizes DTB by abstracting a hydrogen from C6 or C9, generating a dethiobiotinyl carbon radical that is quenched by a sulfide from a [2Fe-2S] (2+) cluster. A similar reaction sequence directed at the other position generates the second C-S bond in the thiophane ring. Since the BS active site holds only one AdoMet and one DTB, it follows that dissociation of methionine and 5'-deoxyadenosine and binding of a second equivalent of AdoMet must be intermediate steps in the formation of biotin. During these dissociation-association steps, a discrete DTB-derived intermediate must remain bound to the enzyme. In this work, we confirm that the conversion of DTB to biotin is accompanied by the reductive cleavage of 2 equiv of AdoMet. A discrepancy between DTB consumption and biotin formation suggests the presence of an intermediate, and we use liquid chromatography and mass spectrometry to demonstrate that this intermediate is indeed 9-mercaptodethiobiotin, generated at approximately 10% of the total enzyme concentration. The amount of intermediate observed is increased when the reaction is run with substoichiometric levels of AdoMet or with the defective enzyme containing the Asn153Ser mutation. The retention of 9-mercaptodethiobiotin as a tightly bound intermediate is consistent with a mechanism involving the stepwise radical-mediated oxidative abstraction of sulfide from an iron-sulfur cluster.     

Contribution of cysteine desulfurase (NifS protein) to the biotin synthase reaction of Escherichia coli

The contribution of cysteine desulfurase, the NifS protein of Klebsiella pneumoniae and the IscS protein of Escherichia coli, to the biotin synthase reaction was investigated in in vitro and in vivo reaction systems with E. coli. When the nifS and nifU genes of K. pneumoniae were coexpressed in E. coli, NifS and NifU proteins in complex (NifU/S complex) and NifU monomer forms were observed. Both the NifU/S complex and the NifU monomer stimulated the biotin synthase reaction in the presence of L-cysteine in an in vitro reaction system. The NifU/S complex enhanced the production of biotin from dethiobiotin by the cells growing in an in vivo reaction system. Moreover, the IscS protein of E. coli stimulated the biotin synthase reaction in the presence of L-cysteine in the cell-free system. These results strongly suggest that cysteine desulfurase participates in the biotin synthase reaction, probably by supplying sulfur to the iron-sulfur cluster of biotin synthase.     

Role of the [2Fe-2S] cluster in recombinant Escherichia coli biotin synthase

Biotin synthase (BioB) converts dethiobiotin into biotin by inserting a sulfur atom between C6 and C9 of dethiobiotin in an S-adenosylmethionine (SAM)-dependent reaction. The as-purified recombinant BioB from Escherichia coli is a homodimeric molecule containing one [2Fe-2S](2+) cluster per monomer. It is inactive in vitro without the addition of exogenous Fe. Anaerobic reconstitution of the as-purified [2Fe-2S]-containing BioB with Fe(2+) and S(2)(-) produces a form of BioB that contains approximately one [2Fe-2S](2+) and one [4Fe-4S](2+) cluster per monomer ([2Fe-2S]/[4Fe-4S] BioB). In the absence of added Fe, the [2Fe-2S]/[4Fe-4S] BioB is active and can produce up to approximately 0.7 equiv of biotin per monomer. To better define the roles of the Fe-S clusters in the BioB reaction, M?ssbauer and electron paramagnetic resonance (EPR) spectroscopy have been used to monitor the states of the Fe-S clusters during the conversion of dethiobiotin to biotin. The results show that the [4Fe-4S](2+) cluster is stable during the reaction and present in the SAM-bound form, supporting the current consensus that the functional role of the [4Fe-4S] cluster is to bind SAM and facilitate the reductive cleavage of SAM to generate the catalytically essential 5'-deoxyadenosyl radical. The results also demonstrate that approximately (2)/(3) of the [2Fe-2S] clusters are degraded by the end of the turnover experiment (24 h at 25 degrees C). A transient species with spectroscopic properties consistent with a [2Fe-2S](+) cluster is observed during turnover, suggesting that the degradation of the [2Fe-2S](2+) cluster is initiated by reduction of the cluster. This observed degradation of the [2Fe-2S] cluster during biotin formation is consistent with the proposed sacrificial S-donating function of the [2Fe-2S] cluster put forth by Jarrett and co-workers (Ugulava et al. (2001) Biochemistry 40, 8352-8358). Interestingly, degradation of the [2Fe-2S](2+) cluster was found not to parallel biotin formation. The initial decay rate of the [2Fe-2S](2+) cluster is about 1 order of magnitude faster than the initial formation rate of biotin, indicating that if the [2Fe-2S] cluster is the immediate S donor for biotin synthesis, insertion of S into dethiobiotin would not be the rate-limiting step. Alternatively, the [2Fe-2S] cluster may not be the immediate S donor. Instead, degradation of the [2Fe-2S] cluster may generate a protein-bound polysulfide or persulfide that serves as the immediate S donor for biotin production.     

Further investigation on the turnover of Escherichia coli biotin synthase with dethiobiotin and 9-mercaptodethiobiotin as substrates

Biotin synthase, a member of the "radical-SAM" family, produces biotin by inserting a sulfur atom between C-6 and C-9 of dethiobiotin. Each of the two saturated carbon atoms is activated through homolytic cleavage of a C-H bond by a deoxyadenosyl radical, issued from the monoelectronic reduction of S-adenosylmethionine (SAM or AdoMet). An important unexplained observation is that the enzyme produces only 1 mol of biotin per enzyme monomer. Some possible reasons for this absence of multiple turnovers are considered here, in connection with the postulated mechanisms. There is a general agreement among several groups that the active form of biotin synthase contains one (4Fe-4S)(2+,1+) center, which mediates the electron transfer to AdoMet, and one (2Fe-2S)(2+) center, which is considered the sulfur source [Ugulava, N. B., Sacanell, C. J., and Jarrett, J. T. (2001) Biochemistry 40, 8352-8358; Tse Sum Bui, B., Benda, R., Schunemann, V., Florentin, D., Trautwein, A. X., and Marquet, A. (2003) Biochemistry 42, 8791-8798; Jameson, G. N. L., Cosper, M. M., Hernandez, H. L., Johnson, M. K., and Huynh, B. H. (2004) Biochemistry 43, 2022-2031]. An alternative hypothesis considers that biotin synthase has a pyridoxal phosphate (PLP)-dependent cysteine desulfurase activity, producing a persulfide which could be the sulfur donor. The absence of turnover was explained by the inhibition due to deoxyadenosine, an end product of the reaction [Ollagnier-de Choudens, S., Mulliez, E., and Fontecave, M. (2002) FEBS Lett. 535, 465-468]. In this work, we show that our purified enzyme has no cysteine desulfurase activity and the required sulfide has to be added as Na(2)S. It cannot be replaced by cysteine, and consistently, PLP has no effect. We observed that deoxyadenosine does not inhibit the reaction either. On the other hand, if the (2Fe-2S)(2+) center is the sulfur source, its depletion after reaction could explain the absence of turnover. We found that after addition of fresh cofactors, including Fe(2+) and S(2)(-), either to the assay when one turn is completed or after purification of the reacted enzyme by different techniques, only a small amount of biotin (0.3-0.4 equiv/monomer) is further produced. This proves that an active enzyme cannot be fully reconstituted after one turn. When 9-mercaptodethiobiotin, which already contains the sulfur atom of biotin, is used as the substrate, the same turnover of one is observed, with similar reaction rates. We postulate that the same intermediate involving the (2Fe-2S) cluster is formed from both substrates, with a rate-determining step following the formation of this intermediate.     

Structure-function studies of Escherichia coli biotin synthase via a chemical modification and site-directed mutagenesis approach.

In Escherichia coli, biotin synthase (bioB gene product) catalyzes the key step in the biotin biosynthetic pathway, converting dethiobiotin (DTB) to biotin. Previous studies have demonstrated that BioB is a homodimer and that each monomer contains an iron-sulfur cluster. The purified BioB protein, however, does not catalyze the formation of biotin in a conventional fashion. The sulfur atom in the iron-sulfur cluster or from the cysteine residues in BioB have been suggested to act as the sulfur donor to form the biotin molecule, and yet unidentified factors were also proposed to be required to regenerate the active enzyme. In order to understand the catalytic mechanism of BioB, we employed an approach involving chemical modification and site-directed mutagenesis. The properties of the modified and mutated BioB species were examined, including DTB binding capability, biotin converting activity, and Fe(2+) content. From our studies, four cysteine residues (Cys 53, 57, 60, and 97) were assigned as the ligands of the iron-sulfur cluster, and Cys to Ala mutations completely abolished biotin formation activity. Two other cysteine residues (Cys 128 and 188) were found to be involved mainly in DTB binding. The tryptophan and histidine residues were suggested to be involved in DTB binding and dimer formation, respectively. The present study also reveals that the iron-sulfur cluster with its ligands are the key components in the formation of the DTB binding site. Based on the current results, a refined model for the reaction mechanism of biotin synthase is proposed.     

MioC is an FMN-binding protein that is essential for Escherichia coli biotin synthase activity in vitro

Biotin synthase is required for the conversion of dethiobiotin to biotin and requires a number of accessory proteins and small molecule cofactors for activity in vitro. We have previously identified two of these proteins as flavodoxin and ferredoxin (flavodoxin) NADP(+) reductase. We now report the identification of MioC as a third essential protein, together with its cloning, purification, and characterization. Purified MioC has a UV-visible spectrum characteristic of a flavoprotein and contains flavin mononucleotide. The presence of flavin mononucleotide and the primary sequence similarity to flavodoxin suggest that MioC may function as an electron transport protein. The role of MioC in the biotin synthase reaction is discussed, and the structure and function of MioC is compared with that of flavodoxin.     

Repression of biotin biosynthesis in Escherichia coli during growth on biotin vitamers.

A strain of Escherichia coli in which the lacZ gene was fused to the bioA promoter was constructed. Colonies of this strain formed Lac(+) colonies on low-biotin agar (1.6 to 4.1 nM) and Lac(-) colonies on high-biotin agar (41 nM). This lac-bio fusion strain was used to study the question of whether cells growing on the biotin vitamers d-biotin-d-sulfoxide (BDS) and dethiobiotin (DTB) generate enough biotin to give maximal repression of beta-galactosidase synthesis. Repression by high concentrations (400 nM) of BDS was almost maximal (about 96%), whereas DTB repression reached a saturation level of about 80% with increasing DTB concentrations. The levels of repression obtained with both vitamers were sufficient to cause the colonies to appear Lac(-). When the lac-bio fusion was transduced into lines carrying mutations (bis) that prevent reduction of BDS to biotin, the transductants were not repressed by added BDS. Repression by BDS is unlikely to result from accumulation of extracellular biotin-related substances because (i) washed bis(+) cells were not detectably derepressed when transferred into medium containing BDS and (ii) washed bis cells were not detectably repressed when transferred into medium in which bis(+) cells had grown. Lactose agar plates containing high concentrations of DTB or BDS comprise an efficient selective medium for bioB or bis mutants and were used to isolate spontaneous mutations of these genes. This method should be adaptable to the selection of mutations in any biosynthetic pathway subject to end-product repression.     

Secondary structure composition of reconstituted subunit b of the Escherichia coli ATP synthase.

Subunit b of the Escherichia coli ATP synthase was isolated by preparative gel electrophoresis, acetone precipitated and after ion-pair extraction redissolved in a buffer either containing n-dodecyl-beta-D-maltoside or sodium cholate. The secondary structure of isolated subunit b was shown to be the same as within the FO complex, but was strongly dependent on the detergent used for replacement of the phospholipid environment. This was shown by an identical tryptic digestion pattern, which was strongly influenced by the detergent used for solubilization. An influence of the detergent n-dodecyl-beta-D-maltoside on the secondary structure of the hydrophilic part of subunit b was also shown for the soluble part of the polypeptide comprising residues Val25 to Leu156 (bsol) using CD spectroscopy. In order to determine the secondary structure of subunit b in its native conformation, isolated subunit b was reconstituted into E. coli lipid vesicles and analyzed with CD spectroscopy. The resulting spectrum revealed a secondary structure composition of 80% alpha helix together with 14% beta turn conformation. These results suggest that subunit b is not a rigid rod-like alpha helix simply linking F1 to FO, but rather provides an inherent flexibility for the storage of elastic energy within the second stalk generated by rotational movements within the F1FO complex.     

Characterization of Escherichia coli B glycogen synthase enzymatic reactions and products.

Previous reports implicate UDPglucose as an active glucosyl donor for the unprimed reaction and “glucoprotein” formation in glycogen biosynthesis in Escherichia coli. Results presented here indicate that UDPglucose and GDPglucose are glucosyl donors in the primed and unprimed reactions catalyzed by purified E. coli B glycogen synthase at less than 5% the rate observed when ADPglucose is the donor. The unprimed reaction is stimulated by 0.25 m citrate and a high molecular weight product is formed similar to that produced when ADPglucose is the glucosyl donor. Physiological amounts of branching enzyme and high concentrations of glycogen inhibit transfer from UDPglucose and GDPglucose. In addition, transfer from UDPglucose is inhibited by ADPglucose. These results strongly suggest that ADPglucose is the physiological donor in both the primed and unprimed reactions. Furthermore, these and previously reported results suggest that one enzyme is involved in the catalysis of the primed, unprimed, and TCA-insoluble product formation reactions. Antiserum prepared against purified E. coli B glycogen synthase inactivates transfer of glucose from either ADPglucose or UDPglucose in the above reactions catalyzed by E. coli B crude extracts. Purified E. coli B glycogen synthase preparations contain significant amounts of α-glucan primer. Evidence shows that this glucan is not covalently attached to the enzyme. Results presented show that formation of material insoluble in TCA and previously considered to be due to “glucoprotein” formation, is in fact due to the generation of long chain length glucan molecules intrinsically acid insoluble. The data suggest that previous results purported to be de novo synthesis of glycogen are due to glucan associated with the glycogen synthase and not to formation of a “glucoprotein” intermediate which then acts as primer for further oligosaccharide synthesis.