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K. V. Rajagopalan - One of the best experts on this subject based on the ideXlab platform.
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In vitro molybdenum ligation to Molybdopterin using purified components.
The Journal of biological chemistry, 2005Co-Authors: Jason D. Nichols, K. V. RajagopalanAbstract: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.
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Structural studies of Molybdopterin synthase provide insights into its catalytic mechanism.
The Journal of biological chemistry, 2003Co-Authors: Michael J. Rudolph, K. V. Rajagopalan, Margot M. Wuebbens, Oliver Turque, Hermann SchindelinAbstract: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.
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Mechanistic and Mutational Studies of Escherichia coli Molybdopterin Synthase Clarify the Final Step of Molybdopterin Biosynthesis
The Journal of biological chemistry, 2003Co-Authors: Margot M. Wuebbens, K. V. RajagopalanAbstract: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.
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Characterization of Escherichia coli MoeB and Its Involvement in the Activation of Molybdopterin Synthase for the Biosynthesis of the Molybdenum Cofactor
The Journal of biological chemistry, 2001Co-Authors: Silke Leimkühler, Margot M. Wuebbens, K. V. RajagopalanAbstract: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.
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Molybdopterin from molybdenum and tungsten enzymes
Advances in Protein Chemistry, 2001Co-Authors: Hermann Schindelin, Caroline Kisker, K. V. RajagopalanAbstract:Publisher Summary This chapter focuses on the structural data describing molybdenum cofactor (Moco)-containing enzymes and discusses their respective catalytic mechanisms. The crystal structures of Moco-containing enzymes have revealed a remarkable degree of structural diversity in the overall polypeptide folds, as well as in the composition and stoichiometry of the Moco. The structures of Moco-containing enzymes representing the four different enzyme families indicate that there is no ancestral Moco-containing enzyme from which all enzymes containing this cofactor is derived. Instead, evolution independently selected at least four different protein folds to harbor the Moco. The structural differences between representatives of the same and different families are analyzed in the chapter. This comparison will show that the Moco-containing enzymes represent a very heterogeneous group with differences in overall enzyme structure, cofactor composition, and stoichiometry, as well as differences in the immediate molybdenum environment. The chapter presents the currently available biochemical and structural data on the enzymes involved in the biosynthesis of Moco.
Silke Leimkühler - One of the best experts on this subject based on the ideXlab platform.
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Reconstitution of Molybdoenzymes with Bis-Molybdopterin Guanine Dinucleotide Cofactors
2019Co-Authors: Paul Kaufmann, Chantal Iobbi-nivol, Silke LeimkühlerAbstract:Molybdoenzymes are ubiquitous, and play important roles in all kingdoms of life. The enzymes' cofactors comprise the metal molybdenum, a special organic ligand system called Molybdopterin (MPT), additional small ligands like water, hydroxide, oxo-, sulfido-or selenido-functions and, in some enzymes, a coordination to the peptide chain of the protein via an amino acid ligand (e.g. serine, aspartate, cysteine or selenosysteine). The so-called molybdenum cofactor (Moco) is deeply buried in the protein at the end of a narrow funnel giving access only to the substrate. In 1974 an assay was developed by Nason and coworkers using the pleotrophic Neurospora crassa mutant nit-1 for the reconstitution of molybdoenzyme activities from crude extracts. These studies lead to the understanding that Moco is the common element in all molybdoenzymes from different organisms. The assay has been further developed since using specific molybdenum enzymes as source of Moco for the reconstitution of diverse purified apo-molybdoenzymes. Alternatively, the molybdenum cofactor can be synthesized in vitro from stable intermediates and can be inserted into apo-molybdoenzymes by the aid of specific Moco-binding chaperones. A general working protocol is described here for the insertion of the bis-Molybdopterin guanine dunucleotide cofactor (bis-MGD) into its target molybdoenzyme using the example of Escherichia coli TMAO reductase. 2
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Reconstitution of Molybdoenzymes with Bis-Molybdopterin Guanine Dinucleotide Cofactors.
Methods in molecular biology (Clifton N.J.), 2018Co-Authors: Paul Kaufmann, Chantal Iobbi-nivol, Silke LeimkühlerAbstract:Molybdoenzymes are ubiquitous and play important roles in all kingdoms of life. The cofactors of these enzymes comprise the metal, molybdenum (Mo), which is bound to a special organic ligand system called Molybdopterin (MPT). Additional small ligands are present at the Mo atom, including water, hydroxide, oxo-, sulfido-, or selenido-functionalities, and in some enzymes, amino acid ligand, such as serine, aspartate, cysteine, or selenocysteine that coordinate the cofactor to the peptide chain of the enzyme. The so-called molybdenum cofactor (Moco) is deeply buried within the protein at the end of a narrow funnel, giving access only to the substrate. In 1974, an assay was developed by Nason and coworkers using the pleiotropic Neurospora crassa mutant, nit-1, for the reconstitution of molybdoenzyme activities from crude extracts. These studies have led to the understanding that Moco is the common element in all molybdoenzymes from different organisms. The assay has been further developed since then by using specific molybdenum enzymes as the source of Moco for the reconstitution of diverse purified apo-molybdoenzymes. Alternatively, the molybdenum cofactor can be synthesized in vitro from stable intermediates and subsequently inserted into apo-molybdoenzymes with the assistance of specific Moco-binding chaperones. A general working protocol is described here for the insertion of the bis-Molybdopterin guanine dinucleotide cofactor (bis-MGD) into its target molybdoenzyme using the example of Escherichia coli trimethylamine N-oxide (TMAO) reductase.
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Molybdopterin dinucleotide biosynthesis in escherichia coli identification of amino acid residues of Molybdopterin dinucleotide transferases that determine specificity for binding of guanine or cytosine nucleotides
Journal of Biological Chemistry, 2011Co-Authors: Meina Neumann, Farida Seduk, Chantal Iobbinivol, Silke LeimkühlerAbstract:The molybdenum cofactor is modified by the addition of GMP or CMP to the C4′ phosphate of Molybdopterin forming the Molybdopterin guanine dinucleotide or Molybdopterin cytosine dinucleotide cofactor, respectively. The two reactions are catalyzed by specific enzymes as follows: the GTP:Molybdopterin guanylyltransferase MobA and the CTP:Molybdopterin cytidylyltransferase MocA. Both enzymes show 22% amino acid sequence identity and are specific for their respective nucleotides. Crystal structure analysis of MobA revealed two conserved motifs in the N-terminal domain of the protein involved in binding of the guanine base. Based on these motifs, we performed site-directed mutagenesis studies to exchange the amino acids to the sequence found in the paralogue MocA. Using a fully defined in vitro system, we showed that the exchange of five amino acids was enough to obtain activity with both GTP and CTP in either MocA or MobA. Exchange of the complete N-terminal domain of each protein resulted in the total inversion of nucleotide specificity activity, showing that the N-terminal domain determines nucleotide recognition and binding. Analysis of protein-protein interactions showed that the C-terminal domain of either MocA or MobA determines the specific binding to the respective acceptor protein.
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Molybdopterin dinucleotide biosynthesis in escherichia coli identificationofaminoacidresiduesofMolybdopterindinucleotide transferasesthatdeterminespecificityforbindingofguanineorcytosine nucleotides
2011Co-Authors: Meina Neumann, Farida Seduk, Chantal Iobbinivol, Silke LeimkühlerAbstract:The molybdenum cofactor is modified by the addition ofGMP or CMP to the C4 (i) formation of precursor Z (3, 4); (ii) formation of MPT fromphosphate of Molybdopterin formingthe Molybdopterin guanine dinucleotide or Molybdopterincytosine dinucleotide cofactor, respectively. The two reactionsare catalyzed by specific enzymes as follows: the GTP:molyb-dopterin guanylyltransferase MobA and the CTP:molybdopt-erin cytidylyltransferase MocA. Both enzymes show 22%amino acid sequence identity and are specific for their respec-tive nucleotides. Crystal structure analysis of MobA revealedtwo conserved motifs in the N-terminal domain of the proteininvolved in binding of the guanine base. Based on these motifs,we performed site-directed mutagenesis studies to exchangethe amino acids to the sequence found in the paralogue MocA.Using a fully defined
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MocA is a specific cytidylyltransferase involved in Molybdopterin cytosine dinucleotide biosynthesis in Escherichia coli.
The Journal of biological chemistry, 2009Co-Authors: Meina Neumann, Farida Seduk, Chantal Iobbi-nivol, Gerd Mittelstädt, Silke LeimkühlerAbstract: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 microm for CTP and 1.17+/-0.18 microm 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.
Ralf R. Mendel - One of the best experts on this subject based on the ideXlab platform.
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Elucidating biosynthetic pathways for vitamins and cofactors.
Natural Product Reports, 2007Co-Authors: Michael E Webb, Ralf R. Mendel, Andrée Marquet, Fabrice Rébeillé, Alison G SmithAbstract:The elucidation of the pathways to the water-soluble vitamins and cofactors has provided many biochemical and chemical challenges. This is a reflection both of their complex chemical nature, and the fact that they are often made in small amounts, making detection of the enzyme activities and intermediates difficult. Here we present an orthogonal review of how these challenges have been overcome using a combination of methods, which are often ingenious. We make particular reference to some recent developments in the study of biotin, pantothenate, folate, pyridoxol, cobalamin, thiamine, riboflavin and Molybdopterin biosynthesis.
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The Mechanism of nucleotide-assisted molybdenum insertion into Molybdopterin. A novel route toward metal cofactor assembly.
The Journal of biological chemistry, 2006Co-Authors: Angel Llamas, Ralf R. Mendel, Tanja Otte, Gerd Multhaup, Guenter SchwarzAbstract:The molybdenum cofactor (Moco) is synthesized by an ancient and conserved biosynthetic pathway. In plants, the two-domain protein Cnx1 catalyzes the insertion of molybdenum into Molybdopterin (MPT), a metal-free phosphorylated pyranopterin carrying an ene-dithiolate. Recently, we identified a novel biosynthetic intermediate, adenylated Molybdopterin (MPT-AMP), which is synthesized by the C-terminal G domain of Cnx1. Here, we show that MPT-AMP and molybdate bind in an equimolar and cooperative way to the other N-terminal E domain (Cnx1E). Tungstate and sulfate compete for molybdate, which demonstrates the presence of an anion-binding site for molybdate. Cnx1E catalyzes the Zn2+-/Mg2+-dependent hydrolysis of MPT-AMP but only when molybdate is bound as co-substrate. MPT-AMP hydrolysis resulted in stoichiometric release of Moco that was quantitatively incorporated into plant apo-sulfite oxidase. Upon Moco formation AMP is release as second product of the reaction. When comparing MPT-AMP hydrolysis with the formation of Moco and AMP a 1.5-fold difference in reaction rates were observed. Together with the strict dependence of the reaction on molybdate the formation of adenylated molybdate as reaction intermediate in the nucleotide-assisted metal transfer reaction to Molybdopterin is proposed.
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Structure of the Molybdopterin-bound Cnx1G domain links molybdenum and copper metabolism.
Nature, 2004Co-Authors: Jochen Kuper, Ralf R. Mendel, Angel Llamas, Hans-jürgen Hecht, Gunter SchwarzAbstract:The molybdenum cofactor is part of the active site of all molybdenum-dependent enzymes, except nitrogenase. The molybdenum cofactor consists of Molybdopterin, a phosphorylated pyranopterin, with an ene-dithiolate coordinating molybdenum. The same pyranopterin-based cofactor is involved in metal coordination of the homologous tungsten-containing enzymes found in archea. The molybdenum cofactor is synthesized by a highly conserved biosynthetic pathway. In plants, the multidomain protein Cnx1 catalyses the insertion of molybdenum into Molybdopterin. The Cnx1 G domain (Cnx1G), whose crystal structure has been determined in its apo form, binds Molybdopterin with high affinity and participates in the catalysis of molybdenum insertion. Here we present two high-resolution crystal structures of Cnx1G in complex with Molybdopterin and with adenylated Molybdopterin (Molybdopterin-AMP), a mechanistically important intermediate. Molybdopterin-AMP is the reaction product of Cnx1G and is subsequently processed in a magnesium-dependent reaction by the amino-terminal E domain of Cnx1 to yield active molybdenum cofactor. The unexpected identification of copper bound to the Molybdopterin dithiolate sulphurs in both structures, coupled with the observed copper inhibition of Cnx1G activity, provides a molecular link between molybdenum and copper metabolism.
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Characterisation of the mob locus of rhodobacter sphaeroides WS8 : MobA is the only gene required for Molybdopterin guanine dinucleotide synthesis
Archives of Microbiology, 2001Co-Authors: Grant Buchanan, Ralf R. Mendel, Gunter Schwarz, Jochen Kuper, Tracy PalmerAbstract:The mob genes of several bacteria have been implicated in the conversion of Molybdopterin to Molybdopterin guanine dinucleotide. The mob locus of Rhodobacter sphaeroides WS8 comprises three genes, mobABC. Chromosomal in-frame deletions in each of the mob genes have been constructed. The mobA mutant strain has inactive DMSO reductase and periplasmic nitrate reductase activities (both Molybdopterin guanine dinucleotide-requiring enzymes), but the activity of xanthine dehydrogenase, a Molybdopterin enzyme, is unaffected. The inability of a mobA mutant to synthesise Molybdopterin guanine dinucleotide is confirmed by analysis of cell extracts of the mobA strain for molybdenum cofactor forms following iodine oxidation. Mutations in mobB and mobC are not impaired for molybdoenzyme activities and accumulate wild-type levels of Molybdopterin and Molybdopterin guanine dinucleotide, indicating they are not compromised in molybdenum cofactor synthesis. In the mobA mutant strain, the inactive DMSO reductase is found in the periplasm, suggesting that molybdenum cofactor insertion is not necessarily a pre-requisite for export.
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Activity of the Molybdopterin-Containing Xanthine Dehydrogenase of Rhodobacter capsulatus Can Be Restored by High Molybdenum Concentrations in a moeA Mutant Defective in Molybdenum Cofactor Biosynthesis
Journal of bacteriology, 1999Co-Authors: Silke Leimkühler, Gunter Schwarz, Ralf R. Mendel, Sieglinde Angermüller, Werner KlippAbstract:During the screening for Rhodobacter capsulatus mutants defective in xanthine degradation, one Tn5 mutant which was able to grow with xanthine as a sole nitrogen source only in the presence of high molybdate concentrations (1 mM), a phenotype resembling Escherichia coli mogA mutants, was identified. Unexpectedly, the corresponding Tn5 insertion was located within the moeA gene. Partial DNA sequence analysis and interposon mutagenesis of regions flanking R. capsulatus moeA revealed that no further genes essential for Molybdopterin biosynthesis are located in the vicinity of moeA and revealed that moeA forms a monocistronic transcriptional unit in R. capsulatus. Amino acid sequence alignments of R. capsulatus MoeA (414 amino acids [aa]) with E. coli MogA (195 aa) showed that MoeA contains an internal domain homologous to MogA, suggesting similar functions of these proteins in the biosynthesis of the molybdenum cofactor. Interposon mutants defective in moeA did not exhibit dimethyl sulfoxide reductase or nitrate reductase activity, which both require the Molybdopterin guanine dinucleotide (MGD) cofactor, even after addition of 1 mM molybdate to the medium. In contrast, the activity of xanthine dehydrogenase, which binds the Molybdopterin (MPT) cofactor, was restored to wild-type levels after the addition of 1 mM molybdate to the growth medium. Analysis of fluorescent derivatives of the molybdenum cofactor of purified xanthine dehydrogenase isolated from moeA and modA mutant strains, respectively, revealed that MPT is inserted into the enzyme only after molybdenum chelation, and both metal chelation and Mo-MPT insertion can occur only under high molybdate concentrations in the absence of MoeA. These data support a model for the biosynthesis of the molybdenum cofactor in which the biosynthesis of MPT and MGD are split at a stage when the molybdenum atom is added to MPT.
Werner Klipp - One of the best experts on this subject based on the ideXlab platform.
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Activity of the Molybdopterin-Containing Xanthine Dehydrogenase of Rhodobacter capsulatus Can Be Restored by High Molybdenum Concentrations in a moeA Mutant Defective in Molybdenum Cofactor Biosynthesis
Journal of bacteriology, 1999Co-Authors: Silke Leimkühler, Gunter Schwarz, Ralf R. Mendel, Sieglinde Angermüller, Werner KlippAbstract:During the screening for Rhodobacter capsulatus mutants defective in xanthine degradation, one Tn5 mutant which was able to grow with xanthine as a sole nitrogen source only in the presence of high molybdate concentrations (1 mM), a phenotype resembling Escherichia coli mogA mutants, was identified. Unexpectedly, the corresponding Tn5 insertion was located within the moeA gene. Partial DNA sequence analysis and interposon mutagenesis of regions flanking R. capsulatus moeA revealed that no further genes essential for Molybdopterin biosynthesis are located in the vicinity of moeA and revealed that moeA forms a monocistronic transcriptional unit in R. capsulatus. Amino acid sequence alignments of R. capsulatus MoeA (414 amino acids [aa]) with E. coli MogA (195 aa) showed that MoeA contains an internal domain homologous to MogA, suggesting similar functions of these proteins in the biosynthesis of the molybdenum cofactor. Interposon mutants defective in moeA did not exhibit dimethyl sulfoxide reductase or nitrate reductase activity, which both require the Molybdopterin guanine dinucleotide (MGD) cofactor, even after addition of 1 mM molybdate to the medium. In contrast, the activity of xanthine dehydrogenase, which binds the Molybdopterin (MPT) cofactor, was restored to wild-type levels after the addition of 1 mM molybdate to the growth medium. Analysis of fluorescent derivatives of the molybdenum cofactor of purified xanthine dehydrogenase isolated from moeA and modA mutant strains, respectively, revealed that MPT is inserted into the enzyme only after molybdenum chelation, and both metal chelation and Mo-MPT insertion can occur only under high molybdate concentrations in the absence of MoeA. These data support a model for the biosynthesis of the molybdenum cofactor in which the biosynthesis of MPT and MGD are split at a stage when the molybdenum atom is added to MPT.
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xanthine dehydrogenase from the phototrophic purple bacterium rhodobacter capsulatus is more similar to its eukaryotic counterparts than to prokaryotic molybdenum enzymes
Molecular Microbiology, 1998Co-Authors: Silke Leimkühler, Monika Kern, Peter S Solomon, Alastair G. Mcewan, Gunter Schwarz, Ralf R. Mendel, Werner KlippAbstract:Fourteen Rhodobacter capsulatus mutants unable to grow with xanthine as sole nitrogen source were isolated by random Tn5 mutagenesis. Five of these Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments hybridizing to oligonucleotides synthesized according to conserved amino acid sequences of eukaryotic xanthine dehydrogenases. DNA sequence analysis of this region revealed two open reading frames, designated xdhA and xdhB, encoding xanthine dehydrogenase. The deduced amino acid sequence of XDHA contains binding sites for two [2Fe-2S] clusters and FAD, whereas XDHB is predicted to contain the Molybdopterin cofactor. In contrast to R. capsulatus, these three cofactor binding sites reside within a single polypeptide chain in eukaryotic xanthine dehydrogenases. The amino acid sequence of xanthine dehydrogenase from R. capsulatus showed a higher degree of similarity to eukaryotic xanthine dehydrogenases than to the xanthine dehydrogenase-related aldehyde oxidoreductase from Desulphovibrio gigas. The expression of an xdhA-lacZ fusion was induced when hypoxanthine or xanthine was added as sole nitrogen source. Mutations in nlfR1 (ntrC) and nifR4 (rpoN, encoding σ) had no influence on xdh gene expression. A putative activator sensing the availability of substrate seems to respond to xanthine but not to hypoxanthine. The transcriptional start site of xdhA was mapped by primer extension analysis. Comparison with known promoter elements revealed no significant homology. Xanthine dehydrogenase from R. capsulatus was purified to homogeneity. The enzyme consists of two subunits with molecular masses of 85 kDa and 50 kDa respectively. N-terminal amino acid sequencing of both subunits confirmed the predicted start codons. The molecular mass of the native enzyme was determined to be 275 kDa, indicating an αβ-subunit structure. Analysis of the molybdenum cofactor of xanthine dehydrogenase from R. capsulatus revealed that it contains the Molybdopterin cofactor and not a Molybdopterin dinucleotide derivative.
Ortwin Meyer - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Xanthine Dehydrogenase from the Anaerobic Bacterium Veillonella atypica and Identification of a Molybdopterin‐Cytosine‐Dinucleotide‐Containing Molybdenum Cofactor
European journal of biochemistry, 1996Co-Authors: Lothar Gremer, Ortwin MeyerAbstract:The molybdenum-containing iron-sulfur flavoprotein xanthine dehydrogenase from the anaerobic bacterium Veillonella atypica has been purified approximately 800-fold with a yield of approximately 40% and a specific activity of approximately 70 μmol ferricyanide reduced · min−1· mg protein−1 with xanthine as electron donor, which corresponds to approximately 30 μmol xanthine oxidized · min−1· mg protein−1 with methylene blue as electron acceptor. The 129-kDa enzyme was a non-covalent heterotrimer with large (82.4 kDa), medium (28.5 kDa) and small (18.4 kDa) subunits. The N-termini of the small and medium polypeptides of V. atypica xanthine dehydrogenase and the corresponding domains of eukaryotic xanthine dehydrogenases were similar, whereas the N-terminus of the large polypeptide was unrelated to eukaryotic xanthine dehydrogenases. The enzyme contained 0.86 atoms Mo, 1.75 atoms Fe, 1.61 atoms acid-labile sulfur and 0.68 molecules FAD/molecule, which corresponds to a 1:2.0:1.9:0.8 molar ratio. Acid hydrolysis revealed 0.95 mol CMP and 0.80 mol AMP/mol xanthine dehydrogenase. After treatment of the enzyme with iodoacetamide, di(carboxamidomethyl)Molybdopterin cytosine dinucleotide was identified, which indicates that Molybdopterin cytosine dinucleotide is the organic portion of the V. atypica xanthine dehydrogenase molybdenum cofactor. The enzyme and its molybdenum cofactor occurred in a 1:1 molar ratio. Xanthine dehydrogenases from eukaryotic sources are characterized by a domain structure and the presence of duplicate copies of two types of [2Fe-2S] clusters. In contrast, the xanthine dehydrogenase from V. atypica had a heterotrimeric subunit structure and a single [2Fe-2S] cluster. In addition, the enzyme indicates the presence of a Molybdopterin dinucleotide as a constituent of a xanthine dehydrogenase molybdenum cofactor.
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characterization of xanthine dehydrogenase from the anaerobic bacterium veillonella atypica and identification of a Molybdopterin cytosine dinucleotide containing molybdenum cofactor
FEBS Journal, 1996Co-Authors: Lothar Gremer, Ortwin MeyerAbstract:The molybdenum-containing iron-sulfur flavoprotein xanthine dehydrogenase from the anaerobic bacterium Veillonella atypica has been purified approximately 800-fold with a yield of approximately 40% and a specific activity of approximately 70 μmol ferricyanide reduced · min−1· mg protein−1 with xanthine as electron donor, which corresponds to approximately 30 μmol xanthine oxidized · min−1· mg protein−1 with methylene blue as electron acceptor. The 129-kDa enzyme was a non-covalent heterotrimer with large (82.4 kDa), medium (28.5 kDa) and small (18.4 kDa) subunits. The N-termini of the small and medium polypeptides of V. atypica xanthine dehydrogenase and the corresponding domains of eukaryotic xanthine dehydrogenases were similar, whereas the N-terminus of the large polypeptide was unrelated to eukaryotic xanthine dehydrogenases. The enzyme contained 0.86 atoms Mo, 1.75 atoms Fe, 1.61 atoms acid-labile sulfur and 0.68 molecules FAD/molecule, which corresponds to a 1:2.0:1.9:0.8 molar ratio. Acid hydrolysis revealed 0.95 mol CMP and 0.80 mol AMP/mol xanthine dehydrogenase. After treatment of the enzyme with iodoacetamide, di(carboxamidomethyl)Molybdopterin cytosine dinucleotide was identified, which indicates that Molybdopterin cytosine dinucleotide is the organic portion of the V. atypica xanthine dehydrogenase molybdenum cofactor. The enzyme and its molybdenum cofactor occurred in a 1:1 molar ratio. Xanthine dehydrogenases from eukaryotic sources are characterized by a domain structure and the presence of duplicate copies of two types of [2Fe-2S] clusters. In contrast, the xanthine dehydrogenase from V. atypica had a heterotrimeric subunit structure and a single [2Fe-2S] cluster. In addition, the enzyme indicates the presence of a Molybdopterin dinucleotide as a constituent of a xanthine dehydrogenase molybdenum cofactor.
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Molybdopterin guanine dinucleotide is the organic moiety of the molybdenum cofactor in respiratory nitrate reductase from pseudomonas stutzeri
Fems Microbiology Letters, 1993Co-Authors: Kurt Frunzke, Ortwin Meyer, Bernd Heiss, Walter G ZumftAbstract:Respiratory nitrate reductase from the denitrifying bacterium Pseudomonas stutzeri is an iron-sulfur enzyme containing the molybdenum cofactor. Hydrolysis of native nitrate reductase with aqueous sulfuric acid revealed 0.92 mol of 5′-GMP per mol of enzyme. The pterin present in the molybdenum cofactor was liberated from the protein and reacted with iodoacetamide. The resulting di(carboxamidomethyl) (cam) derivative was purified on a C18-cartridge and analyzed for its structural elements. Treatment of the cam derivative with nucleotide pyrophosphatase and subsequent HPLC analysis revealed the formation of di(cam)Molybdopterin and 5′-GMP at a 1:1 molar ratio and with a yield of 79% with respect to the molybdenum content of the enzyme. Treatment of the cam derivative with nucleotide pyrophosphatase and alkaline phosphatase led to the liberation of 0.51 mol dephosphodi(cam)Molybdopterin and of 0.59 mol guanosine per mol of enzyme, which is equal to a molar ratio of 1:2.2. The results indicate, that the organic moiety of the molybdenum cofactor of nitrate reductase from P. stutzeri is Molybdopterin guanine dinucleotide of which one mol is contained per mol of nitrate reductase.
