MocA Is a Specific Cytidylyltransferase Involved in Molybdopterin Cytosine Dinucleotide Biosynthesis in Escherichia coli

Abstract

We have purified and characterized a specific CTP:molybdopterin cytidylyltransferase for the biosynthesis of the molybdopterin (MPT) cytosine dinucleotide (MCD) cofactor in Escherichia coli. The protein, named MocA, shows 22% amino acid sequence identity to E. coli MobA, the specific GTP:molybdopterin guanylyltransferase for molybdopterin guanine dinucleotide biosynthesis. MocA is essential for the activity of the MCD-containing enzymes aldehyde oxidoreductase YagTSR and the xanthine dehydrogenases XdhABC and XdhD. Using a fully defined in vitro assay, we showed that MocA, Mo-MPT, CTP, and MgCl2 are required and sufficient for MCD biosynthesis in vitro. The activity of MocA is specific for CTP; other nucleotides such as ATP and GTP were not utilized. In the defined in vitro system a turnover number of 0.37 ± 0.01 min−1 was obtained. A 1:1 binding ratio of MocA to Mo-MPT and CTP was determined to monomeric MocA with dissociation constants of 0.23 ± 0.02 μm for CTP and 1.17 ± 0.18 μm for Mo-MPT. We showed that MocA was also able to convert MPT to MCD in the absence of molybdate, however, with only one catalytic turnover. The addition of molybdate after one turnover gave rise to a higher MCD production, revealing that MCD remains bound to MocA in the absence of molybdate. This work presents the first characterization of a specific enzyme involved in MCD biosynthesis in bacteria. We have purified and characterized a specific CTP:molybdopterin cytidylyltransferase for the biosynthesis of the molybdopterin (MPT) cytosine dinucleotide (MCD) cofactor in Escherichia coli. The protein, named MocA, shows 22% amino acid sequence identity to E. coli MobA, the specific GTP:molybdopterin guanylyltransferase for molybdopterin guanine dinucleotide biosynthesis. MocA is essential for the activity of the MCD-containing enzymes aldehyde oxidoreductase YagTSR and the xanthine dehydrogenases XdhABC and XdhD. Using a fully defined in vitro assay, we showed that MocA, Mo-MPT, CTP, and MgCl2 are required and sufficient for MCD biosynthesis in vitro. The activity of MocA is specific for CTP; other nucleotides such as ATP and GTP were not utilized. In the defined in vitro system a turnover number of 0.37 ± 0.01 min−1 was obtained. A 1:1 binding ratio of MocA to Mo-MPT and CTP was determined to monomeric MocA with dissociation constants of 0.23 ± 0.02 μm for CTP and 1.17 ± 0.18 μm for Mo-MPT. We showed that MocA was also able to convert MPT to MCD in the absence of molybdate, however, with only one catalytic turnover. The addition of molybdate after one turnover gave rise to a higher MCD production, revealing that MCD remains bound to MocA in the absence of molybdate. This work presents the first characterization of a specific enzyme involved in MCD biosynthesis in bacteria. The biosynthesis of the molybdenum cofactor (Moco) 2The abbreviations used are: Mocomolybdenum cofactorMPTmolybdopterinMGDmolybdopterin guanine dinucleotide cofactorMCDmolybdopterin cytosine dinucleotide cofactorhSO-MDhuman sulfite oxidase Moco domainNTAnitrilotriacetic acidFADflavin adenine dinucleotideHPLChigh performance liquid chromatographyXDHxanthine dehydrogenase. 2The abbreviations used are: Mocomolybdenum cofactorMPTmolybdopterinMGDmolybdopterin guanine dinucleotide cofactorMCDmolybdopterin cytosine dinucleotide cofactorhSO-MDhuman sulfite oxidase Moco domainNTAnitrilotriacetic acidFADflavin adenine dinucleotideHPLChigh performance liquid chromatographyXDHxanthine dehydrogenase. is an ancient, ubiquitous, and highly conserved pathway leading to the biochemical activation of molybdenum. In Moco the molybdenum atom is coordinated to the dithiolene group of the 6-alkyl side chain of a pterin called molybdopterin (MPT). Moco biosynthesis has been extensively studied in Escherichia coli by using a combination of biochemical, genetic, and structural approaches (1Rajagopalan K.V. Johnson J.L. J. Biol. Chem. 1992; 267: 10199-10202Abstract Full Text PDF PubMed Google Scholar, 2Rajagopalan K.V. Neidhardt F.C. Escherichia coli and Salmonella. Cellular and Molecular Biology. American Society for Microbiology, Washington, DC1996: 674-679Google Scholar). The biosynthesis of Moco has been divided into four major steps in Escherichia coli: (i) formation of precursor Z (3Hänzelmann P. Schindelin H. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 12870-12875Crossref PubMed Scopus (211) Google Scholar, 4Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 1995; 270: 1082-1087Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), (ii) formation of MPT from precursor Z (5Gutzke G. Fischer B. Mendel R.R. Schwarz G. J. Biol. Chem. 2001; 276: 36268-36274Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 6Pitterle D.M. Johnson J.L. Rajagopalan K.V. J. Biol. Chem. 1993; 268: 13506-13509Abstract Full Text PDF PubMed Google Scholar), (iii) insertion of molybdenum to form Moco via an adenylylated MPT intermediate (7Joshi M.S. Johnson J.L. Rajagopalan K.V. J. Bacteriol. 1996; 178: 4310-4312Crossref PubMed Google Scholar, 8Nichols J.D. Rajagopalan K.V. J. Biol. Chem. 2005; 280: 7817-7822Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 9Nichols J.D. Xiang S. Schindelin H. Rajagopalan K.V. Biochemistry. 2007; 46: 78-86Crossref PubMed Scopus (18) Google Scholar), and (iv) additional modification by covalent addition of GMP to the C4′ phosphate of MPT via a pyrophosphate bond, forming the molybdopterin guanine dinucleotide (MGD) cofactor (10Johnson J.L. Bastian N.R. Rajagopalan K.V. Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 3190-3194Crossref PubMed Scopus (152) Google Scholar, 11Johnson J.L. Indermaur L.W. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 12140-12145Abstract Full Text PDF PubMed Google Scholar). In E. coli, GMP attachment to Moco is catalyzed by the MobA and MobB proteins (12Palmer T. Vasishta A. Whitty P.W. Boxer D.H. Eur. J. Biochem. 1994; 222: 687-692Crossref PubMed Scopus (48) Google Scholar). Although MobA was shown to be essential for this reaction and acts as a GTP:molybdopterin guanylyltransferase (11Johnson J.L. Indermaur L.W. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 12140-12145Abstract Full Text PDF PubMed Google Scholar), the role of MobB still remains uncertain. From the crystal structure, it was postulated that MobB is an adapter protein acting in concert with MobA to achieve the efficient biosynthesis and utilization of MGD (13McLuskey K. Harrison J.A. Schuttelkopf A.W. Boxer D.H. Hunter W.N. J. Biol. Chem. 2003; 278: 23706-23713Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Although MobA was shown to bind MPT, Mo-MPT, and MGD (14Guse A. Stevenson C.E. Kuper J. Buchanan G. Schwarz G. Giordano G. Magalon A. Mendel R.R. Lawson D.M. Palmer T. J. Biol. Chem. 2003; 278: 25302-25307Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), investigations of in vitro studies using purified MobA, MgCl2, GTP, and either MPT or Mo-MPT showed that MGD was only formed by MobA when the molybdenum atom was already ligated to MPT (15Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The formation of bis-MGD is one of the most enigmatic steps in Moco biosynthesis in E. coli. It is still not known whether the two MGD molecules assemble on MobA or instead after the insertion into the respective target proteins like DMSO reductase or nitrate reductase A. In other bacteria like Arthrobacter nicotinovorans, Veillonella atypica, or Oligotropha carboxidovorans, Moco be by the attachment of to the C4′ phosphate of MPT forming the molybdopterin cytosine dinucleotide (MCD) cofactor P. E. PubMed Scopus Google Scholar, Rajagopalan K.V. P. J. Biol. Chem. Full Text PDF PubMed Google Scholar, G. J. Bacteriol. 1995; PubMed Google Scholar). A specific enzyme the CTP:molybdopterin cytidylyltransferase reaction has not been A. and the of a MobA protein for MCD formation was P. E. PubMed Scopus Google however, it was not shown whether the MobA protein was required for MCD biosynthesis or whether it was also involved in the biosynthesis of MGD in this enzymes binding MCD in bacteria an additional modification the molybdenum of a is by a forming or Moco Biochem. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). the MCD-containing protein YagTSR was and characterized in E. coli as a aldehyde oxidoreductase a of using as G. P. S. J. 276: PubMed Scopus Google Scholar). It was shown that for the of an form of the protein was is to be a MCD binding involved in the of the and the insertion of MCD into G. P. S. J. 276: PubMed Scopus Google Scholar). The of the other in E. coli were shown to bind the bis-MGD form of in molybdenum is coordinated to two MGD The other is the protein, the only E. coli protein binding the Mo-MPT form of Moco G. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the role of this protein still remains on YagTSR showed that MCD was into of the of MobA, that a protein is involved in MCD biosynthesis in E. coli G. P. S. J. 276: PubMed Scopus Google Scholar). we the of the specific CTP:molybdopterin we named MocA named by the E. coli of with MocA was shown to the formation of MCD from Mo-MPT and CTP in vitro. we that a in the MCD biosynthesis in E. coli, in an YagTSR protein of have purified and characterized the monomeric MocA protein from E. coli, a protein that 22% sequence identity to the E. coli MobA MocA was shown to the addition of to the C4′ phosphate atom of MPT via a pyrophosphate bond, that the protein is a specific CTP:molybdopterin Using a fully defined in vitro assay, we showed that MocA, Mo-MPT, CTP, and MgCl2 are required and sufficient for MCD biosynthesis in vitro. was able to to in The activity of MocA is specific for CTP as other nucleotides such as ATP or GTP were not by The for CTP was determined to be in the A 1:1 binding ratio of MocA to Mo-MPT and CTP was The binding for the two Mo-MPT and CTP were the for the of CTP in a in the binding for Mo-MPT of the absence of in In the defined in vitro system using purified a turnover number of 0.37 ± 0.01 min−1 was obtained. In a fully defined in vitro system for MGD biosynthesis using E. coli MobA, MPT, GTP, and MgCl2 of MGD only after an for (15Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). in vitro to be for the efficient of either MCD or MGD a turnover The of an protein, like the or a Moco binding the turnover of the reaction S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we not MCD insertion into as was and the was We showed that MocA was also able to convert MPT to MCD in the absence of however, with only one catalytic turnover. The addition of molybdate after one turnover gave rise to a higher MCD production, that remains bound to MocA in the absence of molybdate in a of the We that a catalytic turnover for MocA is only when is This is in with E. coli MobA, it was shown that to MPT has to dinucleotide that molybdate be into MCD when bound to MocA not the that molybdate is after dinucleotide attachment to in vitro of molybdate were required for the of MCD from In this reaction be molybdate by a specific molybdate molybdate the E. coli protein was shown to molybdenum to by MocA it remains to be determined whether also molybdenum to molybdate only a of was determined on MocA in an E. coli this that in the an additional to that MCD is only from Mo-MPT that MPT and that molybdate only into in adenine of in the E. coli and in to the showed that proteins also bind the MCD form of E. coli proteins that bind the aldehyde oxidoreductase YagTSR and the XdhABC and XdhD. characterized MCD-containing enzyme is from It has been that from H. in the absence of molybdate not MCD P. Eur. J. Biochem. PubMed Scopus Google Scholar). In was of MCD or MPT, the protein still of like CTP, or P. Eur. J. Biochem. PubMed Scopus Google Scholar). be by with that H. also a MocA is not by MocA in the absence of molybdate is not into of sequence from that bacteria like or also two for a MocA and a MobA sequence of MocA and MobA proteins from showed conserved proteins The most MocA and MobA are in two conserved the of the The of MobA with bound GTP showed that the binding is the of the protein C.A. Rajagopalan K.V. Schindelin H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). In this MobA proteins a and highly conserved amino acid sequence This sequence is in MocA to a conserved The crystal of MobA also showed that the conserved of a highly conserved is in of the guanine however, to the C.A. Rajagopalan K.V. Schindelin H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). In MocA this amino acid sequence is for a conserved the crystal of MobA with bound MPT or MGD is not it has only been are involved in binding of the MPT MobA and are to and of MPT C.A. Rajagopalan K.V. Schindelin H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). are by in MocA We that conserved amino acid the of MocA and MobA for CTP and GTP, and also MPT and Mo-MPT. studies into the of the two in E. coli. The biosynthesis of the molybdenum cofactor (Moco) 2The abbreviations used are: Mocomolybdenum cofactorMPTmolybdopterinMGDmolybdopterin guanine dinucleotide cofactorMCDmolybdopterin cytosine dinucleotide cofactorhSO-MDhuman sulfite oxidase Moco domainNTAnitrilotriacetic acidFADflavin adenine dinucleotideHPLChigh performance liquid chromatographyXDHxanthine dehydrogenase. 2The abbreviations used are: Mocomolybdenum cofactorMPTmolybdopterinMGDmolybdopterin guanine dinucleotide cofactorMCDmolybdopterin cytosine dinucleotide cofactorhSO-MDhuman sulfite oxidase Moco domainNTAnitrilotriacetic acidFADflavin adenine dinucleotideHPLChigh performance liquid chromatographyXDHxanthine dehydrogenase. is an ancient, ubiquitous, and highly conserved pathway leading to the biochemical activation of molybdenum. In Moco the molybdenum atom is coordinated to the dithiolene group of the 6-alkyl side chain of a pterin called molybdopterin (MPT). Moco biosynthesis has been extensively studied in Escherichia coli by using a combination of biochemical, genetic, and structural approaches (1Rajagopalan K.V. Johnson J.L. J. Biol. Chem. 1992; 267: 10199-10202Abstract Full Text PDF PubMed Google Scholar, 2Rajagopalan K.V. Neidhardt F.C. Escherichia coli and Salmonella. Cellular and Molecular Biology. American Society for Microbiology, Washington, DC1996: 674-679Google Scholar). The biosynthesis of Moco has been divided into four major steps in Escherichia coli: (i) formation of precursor Z (3Hänzelmann P. Schindelin H. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 12870-12875Crossref PubMed Scopus (211) Google Scholar, 4Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 1995; 270: 1082-1087Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), (ii) formation of MPT from precursor Z (5Gutzke G. Fischer B. Mendel R.R. Schwarz G. J. Biol. Chem. 2001; 276: 36268-36274Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 6Pitterle D.M. Johnson J.L. Rajagopalan K.V. J. Biol. Chem. 1993; 268: 13506-13509Abstract Full Text PDF PubMed Google Scholar), (iii) insertion of molybdenum to form Moco via an adenylylated MPT intermediate (7Joshi M.S. Johnson J.L. Rajagopalan K.V. J. Bacteriol. 1996; 178: 4310-4312Crossref PubMed Google Scholar, 8Nichols J.D. Rajagopalan K.V. J. Biol. Chem. 2005; 280: 7817-7822Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 9Nichols J.D. Xiang S. Schindelin H. Rajagopalan K.V. Biochemistry. 2007; 46: 78-86Crossref PubMed Scopus (18) Google Scholar), and (iv) additional modification by covalent addition of GMP to the C4′ phosphate of MPT via a pyrophosphate bond, forming the molybdopterin guanine dinucleotide (MGD) cofactor (10Johnson J.L. Bastian N.R. Rajagopalan K.V. Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 3190-3194Crossref PubMed Scopus (152) Google Scholar, 11Johnson J.L. Indermaur L.W. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 12140-12145Abstract Full Text PDF PubMed Google Scholar). In E. coli, GMP attachment to Moco is catalyzed by the MobA and MobB proteins (12Palmer T. Vasishta A. Whitty P.W. Boxer D.H. Eur. J. Biochem. 1994; 222: 687-692Crossref PubMed Scopus (48) Google Scholar). Although MobA was shown to be essential for this reaction and acts as a GTP:molybdopterin guanylyltransferase (11Johnson J.L. Indermaur L.W. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 12140-12145Abstract Full Text PDF PubMed Google Scholar), the role of MobB still remains uncertain. From the crystal structure, it was postulated that MobB is an adapter protein acting in concert with MobA to achieve the efficient biosynthesis and utilization of MGD (13McLuskey K. Harrison J.A. Schuttelkopf A.W. Boxer D.H. Hunter W.N. J. Biol. Chem. 2003; 278: 23706-23713Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Although MobA was shown to bind MPT, Mo-MPT, and MGD (14Guse A. Stevenson C.E. Kuper J. Buchanan G. Schwarz G. Giordano G. Magalon A. Mendel R.R. Lawson D.M. Palmer T. J. Biol. Chem. 2003; 278: 25302-25307Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), investigations of in vitro studies using purified MobA, MgCl2, GTP, and either MPT or Mo-MPT showed that MGD was only formed by MobA when the molybdenum atom was already ligated to MPT (15Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The formation of bis-MGD is one of the most enigmatic steps in Moco biosynthesis in E. coli. It is still not known whether the two MGD molecules assemble on MobA or instead after the insertion into the respective target proteins like DMSO reductase or nitrate reductase A. In other bacteria like Arthrobacter nicotinovorans, Veillonella atypica, or Oligotropha carboxidovorans, Moco be by the attachment of to the C4′ phosphate of MPT forming the molybdopterin cytosine dinucleotide (MCD) cofactor P. E. PubMed Scopus Google Scholar, Rajagopalan K.V. P. J. Biol. Chem. Full Text PDF PubMed Google Scholar, G. J. Bacteriol. 1995; PubMed Google Scholar). A specific enzyme the CTP:molybdopterin cytidylyltransferase reaction has not been A. and the of a MobA protein for MCD formation was P. E. PubMed Scopus Google however, it was not shown whether the MobA protein was required for MCD biosynthesis or whether it was also involved in the biosynthesis of MGD in this enzymes binding MCD in bacteria an additional modification the molybdenum of a is by a forming or Moco Biochem. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). the MCD-containing protein YagTSR was and characterized in E. coli as a aldehyde oxidoreductase a of using as G. P. S. J. 276: PubMed Scopus Google Scholar). It was shown that for the of an form of the protein was is to be a MCD binding involved in the of the and the insertion of MCD into G. P. S. J. 276: PubMed Scopus Google Scholar). The of the other in E. coli were shown to bind the bis-MGD form of in molybdenum is coordinated to two MGD The other is the protein, the only E. coli protein binding the Mo-MPT form of Moco G. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the role of this protein still remains molybdenum cofactor molybdopterin molybdopterin guanine dinucleotide cofactor molybdopterin cytosine dinucleotide cofactor sulfite oxidase Moco acid adenine dinucleotide performance liquid xanthine dehydrogenase. molybdenum cofactor molybdopterin molybdopterin guanine dinucleotide cofactor molybdopterin cytosine dinucleotide cofactor sulfite oxidase Moco acid adenine dinucleotide performance liquid xanthine dehydrogenase. on YagTSR showed that MCD was into of the of MobA, that a protein is involved in MCD biosynthesis in E. coli G. P. S. J. 276: PubMed Scopus Google Scholar). we the of the specific CTP:molybdopterin we named MocA named by the E. coli of with MocA was shown to the formation of MCD from Mo-MPT and CTP in vitro. we that a in the MCD biosynthesis in E. coli, in an YagTSR protein of have purified and characterized the monomeric MocA protein from E. coli, a protein that 22% sequence identity to the E. coli MobA MocA was shown to the addition of to the C4′ phosphate atom of MPT via a pyrophosphate bond, that the protein is a specific CTP:molybdopterin Using a fully defined in vitro assay, we showed that MocA, Mo-MPT, CTP, and MgCl2 are required and sufficient for MCD biosynthesis in vitro. was able to to in The activity of MocA is specific for CTP as other nucleotides such as ATP or GTP were not by The for CTP was determined to be in the A 1:1 binding ratio of MocA to Mo-MPT and CTP was The binding for the two Mo-MPT and CTP were the for the of CTP in a in the binding for Mo-MPT of the absence of in In the defined in vitro system using purified a turnover number of 0.37 ± 0.01 min−1 was obtained. In a fully defined in vitro system for MGD biosynthesis using E. coli MobA, MPT, GTP, and MgCl2 of MGD only after an for (15Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). in vitro to be for the efficient of either MCD or MGD a turnover The of an protein, like the or a Moco binding the turnover of the reaction S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we not MCD insertion into as was and the was We showed that MocA was also able to convert MPT to MCD in the absence of however, with only one catalytic turnover. The addition of molybdate after one turnover gave rise to a higher MCD production, that remains bound to MocA in the absence of molybdate in a of the We that a catalytic turnover for MocA is only when is This is in with E. coli MobA, it was shown that to MPT has to dinucleotide that molybdate be into MCD when bound to MocA not the that molybdate is after dinucleotide attachment to in vitro of molybdate were required for the of MCD from In this reaction be molybdate by a specific molybdate molybdate the E. coli protein was shown to molybdenum to by MocA it remains to be determined whether also molybdenum to molybdate only a of was determined on MocA in an E. coli this that in the an additional to that MCD is only from Mo-MPT that MPT and that molybdate only into in adenine of in the E. coli and in to the showed that proteins also bind the MCD form of E. coli proteins that bind the aldehyde oxidoreductase YagTSR and the XdhABC and XdhD. characterized MCD-containing enzyme is from It has been that from H. in the absence of molybdate not MCD P. Eur. J. Biochem. PubMed Scopus Google Scholar). In was of MCD or MPT, the protein still of like CTP, or P. Eur. J. Biochem. PubMed Scopus Google Scholar). be by with that H. also a MocA is not by MocA in the absence of molybdate is not into of sequence from that bacteria like or also two for a MocA and a MobA sequence of MocA and MobA proteins from showed conserved proteins The most MocA and MobA are in two conserved the of the The of MobA with bound GTP showed that the binding is the of the protein C.A. Rajagopalan K.V. Schindelin H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). In this MobA proteins a and highly conserved amino acid sequence This sequence is in MocA to a conserved The crystal of MobA also showed that the conserved of a highly conserved is in of the guanine however, to the C.A. Rajagopalan K.V. Schindelin H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). In MocA this amino acid sequence is for a conserved the crystal of MobA with bound MPT or MGD is not it has only been are involved in binding of the MPT MobA and are to and of MPT C.A. Rajagopalan K.V. Schindelin H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). are by in MocA We that conserved amino acid the of MocA and MobA for CTP and GTP, and also MPT and Mo-MPT. studies into the of the two in E. coli. We have purified and characterized the monomeric MocA protein from E. coli, a protein that 22% sequence identity to the E. coli MobA MocA was shown to the addition of to the C4′ phosphate atom of MPT via a pyrophosphate bond, that the protein is a specific CTP:molybdopterin Using a fully defined in vitro assay, we showed that MocA, Mo-MPT, CTP, and MgCl2 are required and sufficient for MCD biosynthesis in vitro. was able to to in The activity of MocA is specific for CTP as other nucleotides such as ATP or GTP were not by The for CTP was determined to be in the A 1:1 binding ratio of MocA to Mo-MPT and CTP was The binding for the two Mo-MPT and CTP were the for the of CTP in a in the binding for Mo-MPT of the absence of in In the defined in vitro system using purified a turnover number of 0.37 ± 0.01 min−1 was obtained. In a fully defined in vitro system for MGD biosynthesis using E. coli MobA, MPT, GTP, and MgCl2 of MGD only after an for (15Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). in vitro to be for the efficient of either MCD or MGD a turnover The of an protein, like the or a Moco binding the turnover of the reaction S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we not MCD insertion into as was and the was We showed that MocA was also able to convert MPT to MCD in the absence of however, with only one catalytic turnover. The addition of molybdate after one turnover gave rise to a higher MCD production, that remains bound to MocA in the absence of molybdate in a of the We that a catalytic turnover for MocA is only when is This is in with E. coli MobA, it was shown that to MPT has to dinucleotide that molybdate be into MCD when bound to MocA not the that molybdate is after dinucleotide attachment to in vitro of molybdate were required for the of MCD from In this reaction be molybdate by a specific molybdate molybdate the E. coli protein was shown to molybdenum to by MocA it remains to be determined whether also molybdenum to molybdate only a of was determined on MocA in an E. coli this that in the an additional to that MCD is only from Mo-MPT that MPT and that molybdate only into The in adenine of in the E. coli and in to the showed that proteins also bind the MCD form of E. coli proteins that bind the aldehyde oxidoreductase YagTSR and the XdhABC and XdhD. characterized MCD-containing enzyme is from It has been that from H. in the absence of molybdate not MCD P. Eur. J. Biochem. PubMed Scopus Google Scholar). In was of MCD or MPT, the protein still of like CTP, or P. Eur. J. Biochem. PubMed Scopus Google Scholar). be by with that H. also a MocA is not by MocA in the absence of molybdate is not into dehydrogenase. of sequence from that bacteria like or also two for a MocA and a MobA sequence of MocA and MobA proteins from showed conserved proteins The most MocA and MobA are in two conserved the of the The of MobA with bound GTP showed that the binding is the of the protein C.A. Rajagopalan K.V. Schindelin H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). In this MobA proteins a and highly conserved amino acid sequence This sequence is in MocA to a conserved The crystal of MobA also showed that the conserved of a highly conserved is in of the guanine however, to the C.A. Rajagopalan K.V. Schindelin H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). In MocA this amino acid sequence is for a conserved the crystal of MobA with bound MPT or MGD is not it has only been are involved in binding of the MPT MobA and are to and of MPT C.A. Rajagopalan K.V. Schindelin H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). are by in MocA We that conserved amino acid the of MocA and MobA for CTP and GTP, and also MPT and Mo-MPT. studies into the of the two in E. coli. We for with the MocA We Rajagopalan for We also for the of with with