Biomedical subjects

K V Rajagopalan

Publications and source records attributed to K V Rajagopalan.

At least 19 recordsLinked to original sources

PubMed2007

Role of the C-terminal Gly-Gly motif of Escherichia coli MoaD, a molybdenum cofactor biosynthesis protein with a ubiquitin fold.

In Escherichia coli, the MoaD protein plays a central role in the conversion of precursor Z to molybdopterin (MPT) during molybdenum cofactor biosynthesis. MoaD has a fold similar to that of ubiquitin and contains a highly conserved C-terminal Gly-Gly motif, which in its active form contains a transferrable sulfur in the form of a thiocarboxylate group. During MPT biosynthesis, MoaD cycles between two different heterotetrameric complexes, one with MoaE to form MPT synthase and the other with MoeB, a protein similar to E1 in the ubiquitin pathway, to regenerate its transferrable sulfur. To determine the specific roles of each of the two terminal Gly residues with regard to the MoaD cycle, variants at the penultimate (Gly80) or terminal (Gly81) residues of both MoaD and thiocarboxylated MoaD were created. These variants were analyzed to determine their effects on complex formation with MoaE and MoeB, formation of the MoaD-acyl-adenylate complex, transfer of sulfur to precursor Z to form MPT, and total cofactor biosynthesis. The combined results show that while conservative substitutions at Gly80 had little effect on any of the processes that were examined, the terminal MoaD residue (Gly81) is important for transfer of sulfur to precursor Z and essential for formation of the MoaD-AMP complex. These results further our understanding of the mechanistic similarities of the MoaD-MoeB reaction to that of the ubiquitin-E1 system.

Amino Acid Motifs↗

PubMed2007

Mutational analysis of Escherichia coli MoeA: two functional activities map to the active site cleft.

The molybdenum cofactor is ubiquitous in nature, and the pathway for Moco biosynthesis is conserved in all three domains of life. Recent work has helped to illuminate one of the most enigmatic steps in Moco biosynthesis, ligation of metal to molybdopterin (the organic component of the cofactor) to form the active cofactor. In Escherichia coli, the MoeA protein mediates ligation of Mo to molybdopterin while the MogA protein enhances this process in an ATP-dependent manner. The X-ray crystal structures for both proteins have been previously described as well as two essential MogA residues, Asp49 and Asp82. Here we describe a detailed mutational analysis of the MoeA protein. Variants of conserved residues at the putative active site of MoeA were analyzed for a loss of function in two different, previously described assays, one employing moeA- crude extracts and the other utilizing a defined system. Oddly, no correlation was observed between the activity in the two assays. In fact, our results showed a general trend toward an inverse relationship between the activity in each assay. Moco binding studies indicated a strong correlation between a variant's ability to bind Moco and its activity in the purified component assay. Crystal structures of the functionally characterized MoeA variants revealed no major structural changes, indicating that the functional differences observed are not due to disruption of the protein structure. On the basis of these results, two different functional areas were assigned to regions at or near the MoeA active site cleft.

Binding Sites↗

PubMed2006

The G473D mutation impairs dimerization and catalysis in human sulfite oxidase.

Among the mutations identified in patients with isolated sulfite oxidase deficiency, the G473D variant is of particular interest since sedimentation analysis reveals that this variant is a monomer, and the importance of the wild-type dimeric state of mammalian sulfite oxidase is not yet well understood. Analysis of recombinant G473D sulfite oxidase indicated that it is severely impaired both in the ability to bind sulfite and in catalysis, with a second-order rate constant 5 orders of magnitude lower than that of the wild type. To elucidate the specific reasons for the severe effects seen in the G473D variant, several other variants were created, including G473A, G473W, and the double mutant R212A/G473D. Despite the inability to form a stable dimer, the G473W variant had 5-fold higher activity than G473D and nearly wild-type activity at pH 7.0 when ferricyanide was the electron acceptor. In contrast, the R212A/G473D variant demonstrated some ability to oligomerize but had undetectable activity. The G473A variant retained the ability to dimerize and had steady-state activity that was comparable to that of the wild type. Furthermore, stopped-flow analysis of the reductive half-reaction of this variant yielded a rate constant nearly 3 times higher than that of the wild type. Examination of the secondary structures of the variants by CD spectroscopy indicated significant random-coil formation in G473D, G473W, and R212A/G473D. These results demonstrate that both the charge and the large size of an Asp residue in this position contribute to the severe effects seen in a patient with the G473D mutation, by causing partial misfolding and monomerization of sulfite oxidase and attenuating both substrate binding and catalytic efficiency during the reaction cycle.

Amino Acid Sequence↗

PubMed2006

High-resolution EXAFS of the active site of human sulfite oxidase: comparison with density functional theory and X-ray crystallographic results.

Much of our knowledge about molybdenum enzymes has originated from EXAFS spectroscopy. This technique provides excellent bond-length accuracy but has only limited bond-length resolution. We have used EXAFS spectroscopy with an extended data range in an attempt to improve bond-length resolution for the molybdenum enzyme sulfite oxidase. The Mo site of sulfite oxidase has two oxygen and three Mo-S ligands (two from cofactor dithiolene plus a cysteine). For the oxidized (Mo(VI)) enzyme, we find that the three Mo-S bond lengths are very similar (within 0.05 A) at 2.41 A, as are the Mo=O ligands at 1.72 A. Density functional theory shows that this is consistent with the proposed active-site structure. The reduced (Mo(IV)) enzyme shows two Mo-S bond lengths at 2.35 A and one at 2.41 A (assigned to cofactor dithiolene and cysteine, respectively, from DFT), together with one Mo=O at 1.72 A and one Mo-OH(2) at 2.30 A.

Binding Sites↗

PubMed2005

The pathogenic human sulfite oxidase mutants G473D and A208D are defective in intramolecular electron transfer.

Mutations G473D and A208D were identified in patients with isolated sulfite oxidase (SO) deficiency, and the equivalent amino acids (G451 and A186, respectively) have been localized to the vicinity of the molybdopterin active site in the X-ray structure of chicken SO [Kisker, C., Schindelin, H., Pacheco, A., Wehbi, W., Garrett, R. M., Rajagopalan, K. V., Enemark, J. H., and Rees, D. C. (1997) Cell 91, 973-983]. To assess the effects of these mutations in human SO, steady-state kinetic studies of enzyme turnover and laser flash photolysis measurements of intramolecular electron transfer (IET) rate constants between the reduced heme [Fe(II)] and Mo(VI) centers were carried out in the recombinant G473D, G473A, G473W, G473D/R212A, and A208D human SO mutants. In the G473D and A208D mutants, the IET rate constants at pH 6.0 are decreased by 3 orders of magnitude relative to that of the wild type. Steady-state kinetic measurements indicate that the IET process is the rate-limiting step in the catalytic cycle of these two mutants. Thus, the large decreases in the IET rate constants and the kcat values, and the large increases in the Km(sulfite) values, rationalize the fatal impact of these mutations. Far-UV CD spectra of G473D indicate that the protein backbone conformation is remarkably changed, and the sedimentation equilibrium indicates that the protein is monomeric. Furthermore, EPR studies also suggest that the active site structure of the Mo(V) form of A208D is different from that of the wild type. In contrast, similar studies on G473A show that it is dimeric, that its Mo(V) active site structure is similar to that of the wild type, and that its IET rate constant is only 2.6-fold smaller than that of the wild type. IET in G473W is severely impaired, and no IET is observed for G473D/R212A. In chicken SO, the equivalent residues (G451 and A186) are both buried inside the protein. Thus, for human SO, the mutations to charged residues at the equivalent sites most likely cause crucial global or localized structural changes, and expose an alternative docking site that may compete with the Mo domain for docking of the heme, thereby retarding IET and efficient catalytic turnover of the sulfite oxidation reaction.

Biopolymers↗

PubMed2005

Structural insights into sulfite oxidase deficiency.

Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.

Amino Acid Sequence↗

PubMed2005

Thermodynamic analysis of subunit interactions in Escherichia coli molybdopterin synthase.

The molybdopterin (MPT) synthase complex in Escherichia coli consists of two MoaE subunits and two MoaD subunits in a heterotetrameric structure with the two MoaE subunits forming a central dimer. Each MoaD subunit binds to a single MoaE molecule to form two identical MoaE/MoaD interfaces. Here we define the thermodynamic properties of the interaction between MoaE and MoaD in MPT synthase using a H/D exchange and matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy based method termed SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX-derived protein folding free energies and m values are reported for MoaE in the presence and absence of MoaD and MoaD-SH, the thiocarboxylated form of MoaD that is essential for the catalytic activity of MPT synthase. The protein folding free energy measurements were used to calculate a dissociation constant of 17 +/- 7 microM for the binding of MoaD to MoaE in inactive MPT synthase and a dissociation constant of 2.6 +/- 0.9 microM for the binding of MoaD-SH to MoaE in active MPT synthase. The increased binding affinity of MoaD-SH for MoaE is consistent with a previously proposed mechanism for the MPT synthase reaction. Using the increased m values exhibited by MoaE in the presence of either MoaD subunit, the solvent accessible surface area buried upon formation of the subunit interface in MPT synthase was estimated to be 2378 A(2) for inactive MPT synthase and 4117 A(2) for active MPT synthase.

Coenzymes↗

PubMed2005

In vitro molybdenum ligation to molybdopterin using purified components.

We have previously shown that Escherichia coli MoeA and MogA are required in vivo for the final step of molybdenum cofactor biosynthesis, the addition of the molybdenum atom to the dithiolene of molybdopterin. MoeA was also shown to facilitate the addition of molybdenum in an assay using crude extracts from E. coli moeA(-) cells. The experiments detailed in this report utilized an in vitro assay for MoeA-mediated molybdenum ligation to de novo synthesized molybdopterin using only purified components and monitoring the reconstitution of human aposulfite oxidase. In this assay, maximum activation was achieved by delaying the addition of aposulfite oxidase to allow for adequate molybdenum coordination to occur. Tungsten, which substitutes for molybdenum in hyperthermophilic organisms, could also be ligated to molybdopterin using this system, though not as efficiently as molybdenum. Addition of thiol compounds to the assay inhibited activity. Addition of MogA also inhibited the reaction. However, in the presence of ATP and magnesium, addition of MogA to the assay increased the level of aposulfite oxidase reconstitution beyond that observed with MoeA alone. This effect was not observed in the absence of MoeA. The results presented here demonstrate that MoeA is responsible for mediating molybdenum ligation to molybdopterin, whereas MogA stimulates this activity in an ATP-dependent manner.

Adenosine Triphosphate↗

PubMed2004

Coordination chemistry at the molybdenum site of sulfite oxidase: redox-induced structural changes in the cysteine 207 to serine mutant.

The redox chemistry of the molybdenum site of the C207S mutant of recombinant human sulfite oxidase has been studied via potentiometric titrations employing both electron paramagnetic resonance (EPR) spectroscopy and X-ray absorption spectroscopy (XAS) as probes of the active site structure. In earlier EXAFS studies, oxidized Cys207Ser enzyme has been shown to possess a novel tri-oxo active site, in which Ser207 does not appear to be a ligand to Mo [George, G. N.; Garrett, R. M.; Prince, R. C.; Rajagopalan, K. V. J. Am. Chem. Soc. 1996, 118, 8588-8592]. Redox titrations show that the active site is modified under reducing conditions to a mono-oxo Mo(IV) species, probably with Ser207 ligated to the metal. The Mo(IV) species can be reoxidized to a mono-oxo Mo(V) species still coordinated to Ser207, which in turn can be further reoxidized to yield the initial tri-oxo Mo(VI) structure with loss of Ser207 ligation.

Binding Sites↗

PubMed2004

Studies on the interaction of NADPH with Rhodobacter sphaeroides biotin sulfoxide reductase.

Rhodobacter sphaeroides biotin sulfoxide reductase (BSOR) contains the bis(molybdopterin guanine dinucleotide)molybdenum cofactor and catalyzes the reduction of D-biotin-D-sulfoxide to biotin. This protein is the only member of the dimethyl sulfoxide reductase family of molybdopterin enzymes that utilizes NADPH as the direct electron donor to the catalytic Mo center. Kinetic studies using stopped-flow spectrophotometry indicate that BSOR reduction by NADPH (>1000 s(-1)) is faster than steady-state turnover (440 s(-1)) and has shown that BSOR reduction occurs in concert with NADPH oxidation with no indication of a Mo(V) intermediate species. Because no crystallographic structure is currently available for BSOR, a protein structure was modeled using the structures for R. sphaeroides dimethyl sulfoxide reductase, Rhodobacter capsulatus dimethyl sulfoxide reductase, and Shewanella massilia trimethylamine N-oxide reductase as the templates. A potential NADPH-binding site was identified and tested by site-directed mutagenesis of residues within the area. Mutation of Arg137 or Asp136 reduced the ability of NADPH to serve as the electron donor to BSOR, indicating that the NADPH-binding site in BSOR is located in the active-site funnel of the putative structure where it can directly reduce the Mo center. Along with kinetic and spectroscopic data, the location of this binding site supports a direct hydride transfer mechanism for NADPH reduction of BSOR.

Amino Acid Sequence↗

PubMed2004

Evidence for the physiological role of a rhodanese-like protein for the biosynthesis of the molybdenum cofactor in humans.

Recent studies have identified the human genes involved in the biosynthesis of the molybdenum cofactor. The human MOCS3 protein contains an N-terminal domain similar to the Escherichia coli MoeB protein and a C-terminal segment displaying similarities to the sulfurtransferase rhodanese. The MOCS3 protein is believed to catalyze both the adenylation and the subsequent generation of a thiocarboxylate group at the C terminus of the smaller subunit of molybdopterin (MPT) synthase. The MOCS3 rhodanese-like domain (MOCS3-RLD) was purified after heterologous expression in E. coli and was shown to catalyze the transfer of sulfur from thiosulfate to cyanide. In a defined in vitro system for the generation of MPT from precursor Z, the sulfurated form of MOCS3-RLD was able to provide the sulfur for the thiocarboxylation of MOCS2A, the small MPT synthase subunit in humans. Mutation of the putative persulfide-forming active-site cysteine residue C412 abolished the sulfurtransferase activity of MOCS3-RLD completely, showing the importance of this cysteine residue for catalysis. In contrast to other mammalian rhodaneses, which are mostly localized within mitochondria, MOCS3 in addition to the subunits of MPT synthase are localized in the cytosol.

Base Sequence↗

PubMed2004

The role of tyrosine 343 in substrate binding and catalysis by human sulfite oxidase.

In the crystal structure of chicken sulfite oxidase, the residue Tyr(322) (Tyr(343) in human sulfite oxidase) was found to directly interact with a bound sulfate molecule and was proposed to have an important role in mediating the substrate specificity and catalytic activity of this molybdoprotein. In order to understand the role of this residue in the catalytic mechanism of sulfite oxidase, steady-state and stopped-flow analyses were performed on wild-type and Y343F human sulfite oxidase over the pH range 6-10. In steady-state assays of Y343F sulfite oxidase using cytochrome c as the electron acceptor, k(cat) was somewhat impaired ( approximately 34% wild-type activity at pH 8.5), whereas the K(m)(sulfite) showed a 5-fold increase over wild type. In rapid kinetic assays of the reductive half-reaction of wild-type human sulfite oxidase, k(red)(heme) changed very little over the entire pH range, with a significant increase in K(d)(sulfite) at high pH. The k(red)(heme) of the Y343F variant was significantly impaired across the entire pH range, and unlike the wild-type protein, both k(red)(heme) and K(d)(sulfite) were dependent on pH, with a significant increase in both kinetic parameters at high pH. Additionally, reduction of the molybdenum center by sulfite was directly measured for the first time in rapid reaction assays using sulfite oxidase lacking the N-terminal heme-containing domain. Reduction of the molybdenum center was quite fast (k(red)(Mo) = 972 s(-1) at pH 8.65 for wild-type protein), indicating that this is not the rate-limiting step in the catalytic cycle. Reduction of the molybdenum center of the Y343F variant by sulfite was more significantly impaired at high pH than at low pH. These results demonstrate that the Tyr(343) residue is important for both substrate binding and oxidation of sulfite by sulfite oxidase.

Animals↗

PubMed2003

Essential role of conserved arginine 160 in intramolecular electron transfer in human sulfite oxidase.

Arginine 160 in human sulfite oxidase (SO) is conserved in all SO species sequenced to date. Previous steady-state kinetic studies of the R160Q human SO mutant showed a remarkable decrease in k(cat)/K(m)(sulfite) of nearly 1000-fold, which suggests that Arg 160 in human SO makes an important contribution to the binding of sulfite near the molybdenum cofactor [Garrett, R. M., Johnson, J. L., Graf, T. N., Feigenbaum, A., Rajagopalan, K. V. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6394-6398]. In the crystal structure of chicken SO, Arg 138, the equivalent of Arg 160 in human SO, is involved in the formation of a positively charged sulfite binding site [Kisker, C., Schindelin, H., Pacheco, A., Wehbi, W., Garnett, R. M., Rajagopalan, K. V., Enemark, J. H., Rees, D. C. (1997) Cell 91, 973-983]. To further assess the role of Arg 160 in human SO, intramolecular electron transfer (IET) rates between the reduced heme [Fe(II)] and oxidized molybdenum [Mo(VI)] centers in the wild type, R160Q, and R160K human SO forms were investigated by laser flash photolysis. In the R160Q mutant, the IET rate constant at pH 6.0 was decreased by nearly 3 orders of magnitude relative to wild type, which indicates that the positive charge of Arg 160 is essential for efficient IET in human SO. Furthermore, the IET rate constant for the R160K mutant is about one-fourth that of the wild type enzyme, which strongly indicates that it is the loss of charge of Arg 160, and not its precise location, that is responsible for the much larger decrease in IET rates in the R160Q mutant. Steady-state kinetic measurements indicate that IET is rate-limiting in the catalytic cycle of the R160Q mutant. Thus, the large decrease in the IET rate constant rationalizes the fatal impact of this mutation in patients with this genetic disorder.

Animals↗

PubMed2003

The 1.2 A structure of the human sulfite oxidase cytochrome b(5) domain.

The molybdenum- and iron-containing enzyme sulfite oxidase catalyzes the physiologically vital oxidation of sulfite to sulfate. Sulfite oxidase contains three domains: an N-terminal cytochrome b(5) domain, a central domain harboring the molybdenum cofactor (Moco) and a C-terminal dimerization domain. Oxidation of the substrate sulfite is coupled to the transfer of two electrons to the molybdenum cofactor. Subsequently, these electrons are passed on, one at a time, to the b(5) heme of sulfite oxidase and from there to the soluble electron carrier cytochrome c. The crystal structure of the oxidized human sulfite oxidase cytochrome b(5) domain has been determined at 1.2 A resolution and has been refined to a crystallographic R factor of 0.107 (R(free) = 0.137). A comparison of this structure with other b(5)-type cytochromes reveals distinct structural features present in the sulfite oxidase b(5) domain which promote optimal electron transport between the Moco of sulfite oxidase and the heme of cytochrome c.

Amino Acid Sequence↗

PubMed2003

Mechanistic studies of human molybdopterin synthase reaction and characterization of mutants identified in group B patients of molybdenum cofactor deficiency.

Biosynthesis of the molybdenum cofactor involves the initial formation of precursor Z, its subsequent conversion to molybdopterin (MPT) by MPT synthase, and attachment of molybdenum to the dithiolene moiety of MPT. The sulfur used for the formation of the dithiolene group of MPT exists in the form of a thiocarboxylate group at the C terminus of the smaller subunit of MPT synthase. Human MPT synthase contains the MOCS2A and MOCS2B proteins that display homology to the Escherichia coli proteins MoaD and MoaE, respectively. MOCS2A and MOCS2B were purified after heterologous expression in E. coli, and the separately purified subunits readily assemble into a functional MPT synthase tetramer. The rate of conversion of precursor Z to MPT by the human enzyme is slower than that of the eubacterial homologue. To obtain insights into the molecular mechanism leading to human molybdenum cofactor deficiency, site-specific mutations identified in patients showing symptoms of molybdenum cofactor deficiency were generated. Characterization of a V7F substitution in MOCS2A, identified in a patient with an unusual mild form of the disease, showed that the mutation weakens the interaction between MOCS2A and MOCS2B, whereas a MOCS2B-E168K mutation identified in a severely affected patient attenuates binding of precursor Z.

Amino Acid Sequence↗

PubMed2003

Recombinant Rhodobacter capsulatus xanthine dehydrogenase, a useful model system for the characterization of protein variants leading to xanthinuria I in humans.

Rhodobacter capsulatus xanthine dehydrogenase (XDH) forms an (alphabeta)2 heterotetramer and is highly homologous to homodimeric eukaryotic XDHs. The crystal structures of bovine XDH and R. capsulatus XDH showed that the two proteins have highly similar folds. We have developed an efficient system for the recombinant expression of R. capsulatus XDH in Escherichia coli. The recombinant protein shows spectral features and a range of substrate specificities similar to bovine milk xanthine oxidase. However, R. capsulatus XDH is at least 5 times more active than bovine XDH and, unlike mammalian XDH, does not undergo the conversion to the oxidase form. EPR spectra were obtained for the FeS centers of the enzyme showing an axial signal for FeSI, which is different from that reported for xanthine oxidase. X-ray absorption spectroscopy at the iron and molybdenum K-edge and the tungsten LIII-edge have been used to probe the different metal coordinations of variant forms of the enzyme. Based on a mutation identified in a patient suffering from xanthinuria I, the corresponding arginine 135 was substituted to a cysteine in R. capsulatus XDH, and the protein variant was purified and characterized. Two different forms of XDH-R135C were purified, an active (alphabeta)2 heterotetrameric form and an inactive (alphabeta) heterodimeric form. The active form contains a full complement of redox centers, whereas in the inactive form the FeSI center is likely to be missing.

Absorptiometry, Photon↗

PubMed2003

Structural studies of molybdopterin synthase provide insights into its catalytic mechanism.

Molybdenum cofactor biosynthesis is an evolutionarily conserved pathway present in eubacteria, archaea, and eukaryotes, including humans. Genetic deficiencies of enzymes involved in cofactor biosynthesis in humans lead to a severe and usually fatal disease. The molybdenum cofactor contains a tricyclic pyranopterin, termed molybdopterin, that bears the cis-dithiolene group responsible for molybdenum ligation. The dithiolene group of molybdopterin is generated by molybdopterin synthase, which consists of a large (MoaE) and small (MoaD) subunit. The crystal structure of molybdopterin synthase revealed a heterotetrameric enzyme in which the C terminus of each MoaD subunit is deeply inserted into a MoaE subunit to form the active site. In the activated form of the enzyme, the MoaD C terminus is present as a thiocarboxylate. The present study identified the position of the thiocarboxylate sulfur by exploiting the anomalous signal originating from the sulfur atom. The structure of molybdopterin synthase in a novel crystal form revealed a binding pocket for the terminal phosphate of molybdopterin, the product of the enzyme, and suggested a binding site for the pterin moiety present in precursor Z and molybdopterin. Finally, the crystal structure of the MoaE homodimer provides insights into the conformational changes accompanying binding of the MoaD subunit.

Binding Sites↗

PubMed2003

Mechanistic and mutational studies of Escherichia coli molybdopterin synthase clarify the final step of molybdopterin biosynthesis.

Biosynthesis of the molybdenum cofactor, a chelate of molybdenum or tungsten with a novel pterin, occurs in virtually all organisms including humans. In the cofactor, the metal is complexed to the unique cis-dithiolene moiety located on the pyran ring of molybdopterin. Escherichia coli molybdopterin synthase, the protein responsible for adding the dithiolene to a desulfo precursor termed precursor Z, is a dimer of dimers containing the MoaD and MoaE proteins. The sulfur used for dithiolene formation is carried in the form of a thiocarboxylate at the MoaD C terminus. Using an intein expression system for preparation of thiocarboxylated MoaD, the mechanism of the molybdopterin synthase reaction was examined. A stoichiometry of 2 molecules of thiocarboxylated MoaD per conversion of a single precursor Z molecule to molybdopterin was observed. Examination of several synthase variants bearing mutations in the MoaE subunit identified Lys-119 as a residue essential for activity and Arg-39 and Lys-126 as other residues critical for the reaction. An intermediate of the synthase reaction was identified and characterized. This intermediate remains tightly associated with the protein and is the predominant product formed by synthase containing the K126A variant of MoaE. Mass spectral data obtained from protein-bound intermediate are consistent with a monosulfurated structure that contains a terminal phosphate group similar to that present in molybdopterin.

Chromatography↗