molybdenum-cofactor and molybdopterin-guanine-dinucleotide

The well-studied enterobacterium Escherichia coli present in the human gut can reduce trimethylamine N-oxide (TMAO) to trimethylamine during anaerobic respiration. The TMAO reductase TorA is a monomeric, bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor-containing enzyme that belongs to the dimethyl sulfoxide reductase family of molybdoenzymes. We report on a system for the in vitro reconstitution of TorA with molybdenum cofactors (Moco) from different sources. Higher TMAO reductase activities for TorA were obtained when using Moco sources containing a sulfido ligand at the molybdenum atom. For the first time, we were able to isolate functional bis-MGD from Rhodobacter capsulatus formate dehydrogenase (FDH), which remained intact in its isolated state and after insertion into apo-TorA yielded a highly active enzyme. Combined characterizations of the reconstituted TorA enzymes by electron paramagnetic resonance spectroscopy and direct electrochemistry emphasize that TorA activity can be modified by changes in the Mo coordination sphere. The combination of these results together with studies of amino acid exchanges at the active site led us to propose a novel model for binding of the substrate to the molybdenum atom of TorA.

Topics: Coenzymes; Cytochrome P-450 Enzyme System; Escherichia coli; Escherichia coli Proteins; Guanine Nucleotides; Humans; Metalloproteins; Models, Molecular; Molybdenum; Molybdenum Cofactors; Oxidoreductases, N-Demethylating; Pteridines; Pterins; Sulfides

The molybdenum cofactor (Moco) exists in different variants in the cell and can be directly inserted into molybdoenzymes utilizing the molybdopterin (MPT) form of Moco. In bacteria such as Rhodobacter capsulatus and Escherichia coli, MPT is further modified by attachment of a GMP nucleotide, forming MPT guanine dinucleotide (MGD). In this work, we analyzed the distribution and targeting of different forms of Moco to their respective user enzymes by proteins that bind Moco and are involved in its further modification. The R. capsulatus proteins MogA, MoeA, MobA, and XdhC were purified, and their specific interactions were analyzed. Interactions between the protein pairs MogA-MoeA, MoeA-XdhC, MoeA-MobA, and XdhC-MobA were identified by surface plasmon resonance measurements. In addition, the transfer of Moco produced by the MogA-MoeA complex to XdhC was investigated. A direct competition of MobA and XdhC for Moco binding was determined. In vitro analyses showed that XdhC bound to MobA, prevented the binding of Moco to MobA, and thereby inhibited MGD biosynthesis. The data were confirmed by in vivo studies in R. capsulatus cells showing that overproduction of XdhC resulted in a 50% decrease in the activity of bis-MGD-containing Me(2)SO reductase. We propose that, in bacteria, the distribution of Moco in the cell and targeting to the respective user enzymes are accomplished by specific proteins involved in Moco binding and modification.

Topics: Carrier Proteins; Coenzymes; Escherichia coli; Escherichia coli Proteins; Guanine Nucleotides; Metalloproteins; Molybdenum Cofactors; Protein Binding; Pteridines; Pterins; Recombinant Proteins; Rhodobacter capsulatus; Sulfurtransferases

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

Topics: Bacteriological Techniques; Binding, Competitive; Chromatography, High Pressure Liquid; Coenzymes; Deuterium; Diethyl Pyrocarbonate; Enzyme Activation; Guanine Nucleotides; Kinetics; Metalloproteins; Molybdenum Cofactors; NADP; Oxidoreductases; Potassium Cyanide; Pteridines; Pterins; Recombinant Proteins; Rhodobacter sphaeroides; Substrate Specificity; Tungsten

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.

Topics: Cloning, Molecular; Coenzymes; Gene Deletion; Gene Expression Regulation, Bacterial; Genes, Bacterial; Guanine Nucleotides; Iodine; Iron-Sulfur Proteins; Metalloproteins; Molybdenum Cofactors; Nitrate Reductase; Nitrate Reductases; Oxidoreductases; Periplasm; Pteridines; Pterins; Rhodobacter sphaeroides; Xanthine Dehydrogenase

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

Topics: Alanine; Amino Acid Sequence; Amino Acids; Binding Sites; Biotin; Catalysis; Chromatography, High Pressure Liquid; Coenzymes; Conserved Sequence; Cysteine; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Factor Xa; Glutathione Transferase; Guanine Nucleotides; Iron-Sulfur Proteins; Ligands; Mass Spectrometry; Metalloproteins; Molecular Sequence Data; Molybdenum Cofactors; Mutagenesis, Site-Directed; NADP; Oxidative Stress; Oxidoreductases; Oxygen; Paraquat; Pteridines; Pterins; Recombinant Fusion Proteins; Rhodobacter sphaeroides; Sequence Homology, Amino Acid; Serine; Spectrometry, Fluorescence; Threonine; Time Factors

The requirement of MobA for molybdoenzymes with different molybdenum cofactors was analyzed in Rhodobacter capsulatus. MobA is essential for DMSO reductase and nitrate reductase activity, both enzymes containing the molybdopterin guanine dinucleotide cofactor (MGD), but not for active xanthine dehydrogenase, harboring the molybdopterin cofactor. In contrast to the mob locus of Escherichia coli and R. sphaeroides, the mobB gene is not located downstream of mobA in R. capsulatus. The mobA gene is expressed constitutively at low levels and no increase in mobA expression could be observed even under conditions of high MGD demand.

Topics: Bacterial Proteins; Blotting, Southern; Chromosome Mapping; Coenzymes; DNA, Bacterial; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Guanine Nucleotides; Iron-Sulfur Proteins; Metalloproteins; Molecular Sequence Data; Molybdenum; Molybdenum Cofactors; Mutagenesis, Insertional; Nitrate Reductase; Nitrate Reductases; Oxidoreductases; Pteridines; Pterins; Rhodobacter capsulatus; Sulfurtransferases; Xanthine Dehydrogenase

Analysis of dimethylsulfoxide reductase from Rhodobacter capsulatus showed that it contained 1 mol Mo and 2 mol GMP. This indicates that the molybdenum cofactor in dimethylsulfoxide reductase is bis(molybdopterin guanine dinucleotide) molybdenum. The absorption spectrum of the molybdopterin guanine dinucleotide released from dimethylsulfoxide reductase after denaturation of the holoenzyme was compared with those of pterin standards of known redox state. The spectra were most similar to pterin standards in the dihydro state and oxidised state. The reduction of 2,6-dichloroindophenol by molybdopterin guanine dinucleotide released from dimethylsulfoxide reductase and by pterin standards was also measured and approximately 2 mol electrons/2 mol molybdopterin guanine dinucleotide were found to reduce 2,6-dichloroindophenol. These results are consistent with the presence of one molybdopterin guanine dinucleotide moiety with a pyrazine ring at the oxidation level of a dihydropteridine and one molybdopterin guanine dinucleotide moiety with a pyrazine ring at the oxidation level of a fully aromatic pteridine. It is suggested that the pyrazine ring of Q-molybdopterin guanine dinucleotide is fully aromatic and contains a 5,6 double bond.

Topics: 2,6-Dichloroindophenol; Bacterial Proteins; Coenzymes; Electron Transport; Guanine Nucleotides; Guanosine Monophosphate; Iron-Sulfur Proteins; Metalloproteins; Molecular Structure; Molybdenum; Molybdenum Cofactors; Organometallic Compounds; Oxidation-Reduction; Oxidoreductases; Phosphates; Protein Denaturation; Pteridines; Pterins; Rhodobacter capsulatus; Spectrophotometry

The molybdenum-containing iron-sulfur protein 1,2,3,5-tetrahydroxybenzene: 1,2,3-trihydroxybenzene hydroxyltransferase (transhydroxylase) of Pelobacter acidigallici was investigated by various techniques including mass spectrometry and electron paramagnetic resonance. Mass spectrometry confirmed that the 133-kDa protein is a heterodimer consisting of an alpha subunit (100.4 kDa) and a beta subunit (31.3 kDa). The presence of a molybdenum cofactor was documented by fluorimetric analysis of the oxidized form A of molybdopterin. The enzyme contained 1.55 +/- 0.14 mol pterin and 0.92 +/- 0.25 mol molybdenum/mol enzyme (133 kDa). Alkylation of the molybdenum cofactor with iodoacetamide formed di(carboxamidomethyl)-molybdopterin. Upon acid hydrolysis, 1.4 mol 5'GMP/mol enzyme (133 kDa) was released indicating that molybdenum is bound by a molybdopterin guanine dinucleotide. The alpha and beta subunits were separated by preparative gel electrophoresis. Both subunit fractions were free of molybdenum but contained equal amounts of a fluorescent form of the molybdenum cofactors. Mass spectrometry at various pH values revealed that an acid-labile cofactor was released from the large subunit and also from the small subunit. At X-band, 5-25 K, transhydroxylase (as isolated) showed minor EPR resonances with apparent g values around 4.3, 2.03 and, depending on the preparation, a further signal at g of approximately 1.98. This signal was still detectable above 70 K and was attributed to a Mo(V) center. Upon addition of dithionite, a complex set of intense resonances appeared in the region g 2.08-1.88. From their temperature dependence, three distinct sites could be identified: the Fe-S center I with gx,y,z at approximately 1.875, 1.942 and 2.087 (gav 1.968, detectable < 20 K); the Fe-S center II with gx,y,z at approximately 1.872, 1.955 and 2.051 (gav 1.959, detectable > 20 K); and the Mo(V) center consisting of a multiple signal around g 1.98 (detectable > 70 K).

Topics: Amino Acid Sequence; Bacteria, Anaerobic; Binding Sites; Coenzymes; Electron Spin Resonance Spectroscopy; Guanine Nucleotides; Iron-Sulfur Proteins; Mass Spectrometry; Metalloproteins; Mixed Function Oxygenases; Molecular Sequence Data; Molecular Structure; Molecular Weight; Molybdenum; Molybdenum Cofactors; Protein Conformation; Pteridines; Pterins

The periplasmic dimethyl sulfoxide reductase (DMSOR) from the photosynthetic purple bacterium Rhodobacter capsulatus functions as the terminal electron acceptor in its respiratory chain. The enzyme catalyzes the reduction of highly oxidized substrates like dimethyl sulfoxide to dimethyl sulfide. At a molybdenum redox center, two single electrons are transferred from cytochrome C556 to the substrate dimethyl sulfoxide, generating dimethyl sulfide and (with two protons) water. The enzyme was purified and crystallized in space group P4(1)2(1)2 with unit cell dimensions of a = b = 80.7 A and c = 229.2 A. The crystals diffract beyond 1.8 A with synchrotron radiation. The three-dimensional structure was solved by a combination of multiple isomorphous replacement and molecular replacement techniques. The atomic model was refined to an R-factor of 0.169 for 57,394 independent reflections. The spherical protein consists of four domains with a funnel-like cavity that leads to the freely accessible metal-ion redox center. The bis(molybdopterin guanine dinucleotide) molybdenum cofactor (1541 Da) of the single chain protein (85,033 Da) has the molybdenum ion bound to the cis-dithiolene group of only one molybdopterin guanine dinucleotide molecule. Three additional ligands, two oxo groups and the oxygen of a serine side-chain, are bound to the molybdenum ion. The second molybdopterin system is not part of the ligand sphere of the metal center with its sulfur atoms at distances of 3.5 A and 3.8 A away. It might be involved in electron shuttling from the protein surface to the molybdenum center.

Topics: Amino Acid Sequence; Binding Sites; Coenzymes; Computer Simulation; Crystallography, X-Ray; Guanine Nucleotides; Iron-Sulfur Proteins; Metalloproteins; Models, Molecular; Molecular Sequence Data; Molybdenum Cofactors; Oxidoreductases; Protein Structure, Tertiary; Pteridines; Pterins; Rhodobacter capsulatus

All known molybdoenzymes other than nitrogenase contain the metal in association with molybdopterin or one of its dinucleotide variants. All eukaryotic molybdoproteins have been found to contain only molybdopterin, whereas the majority of bacterial enzymes contain one or another of the dinucleotides of molybdopterin. In contrast, xanthine dehydrogenase from Pseudomonas aeruginosa contains molybdopterin rather than a dinucleotide. To examine whether P. aeruginosa contains any dinucleotide of molybdopterin, cells were subjected to an analytical procedure which converts molybdopterin variants to the highly fluorescent Form A derivatives. The results showed that P. aeruginosa cells do contain molybdopterin guanine dinucleotide. The same procedure showed that rat liver does not contain any of the dinucleotides of molybdopterin.

Topics: Animals; Chromatography, Ion Exchange; Coenzymes; Guanine Nucleotides; Liver; Metalloproteins; Molybdenum Cofactors; Pseudomonas aeruginosa; Pteridines; Pterins; Rats; Xanthine Dehydrogenase

The chlorate-resistant mutants of Escherichia coli are affected in the biosynthesis of the molybdenum cofactor and show pleiotropic loss of the activities of those enzymes which require the cofactor. The molybdenum cofactor in all molybdoenzymes other than nitrogenase is a complex of the metal with a unique pterin termed molybdopterin. The molybdenum cofactor in a number of E. coli enzymes has been shown to contain GMP in addition to the metal-molybdopterin complex, with the GMP appended in pyrophosphate linkage to the terminal phosphate ester on the molybdopterin side chain. In this paper, we have examined the biochemistry of the chlB mutant and show that the gene product of the chlB locus is essential for the addition of the GMP moiety to form molybdopterin guanine dinucleotide, a step which occurs late in the cofactor biosynthetic pathway in E. coli. Sensitive techniques were developed for the identification of fluorescent derivatives of molybdopterin and of molybdopterin guanine dinucleotide in extracts of E. coli cells. Wild type cells were shown to contain both molybdopterin and molybdopterin guanine dinucleotide, while cells of chlB mutants were found to contain elevated levels of molybdopterin but no detectable molybdopterin guanine dinucleotide.

Topics: Chromatography, High Pressure Liquid; Coenzymes; Escherichia coli; Fluorescence Polarization; Guanine Nucleotides; Guanosine Monophosphate; Metalloproteins; Molybdenum Cofactors; Mutation; Pteridines; Pterins; Pyrophosphatases

The nature of molybdenum cofactor in the bacterial enzyme dimethyl sulfoxide reductase has been investigated by application of alkylation conditions that convert the molybdenum cofactor in chicken liver sulfite oxidase and milk xanthine oxidase to the stable, well-characterized derivative [di(carboxamidomethyl)]molybdopterin. The alkylated pterin obtained from dimethyl sulfoxide reductase was shown to be a modified form of alkylated molybdopterin with increased absorption in the 250-nm region of the spectrum and altered chromatographic behavior. The complex alkylated pterin was resolved into two components by treatment with nucleotide pyrophosphatase. These were identified as di(carboxamidomethyl)molybdopterin and GMP by their absorption spectra, coelution with standard compounds, and by further degradation by alkaline phosphatase to dephospho [di(carboxamidomethyl)]molybdopterin and guanosine. The GMP moiety was sensitive to periodate, identifying it as the 5' isomer. Chemical analysis of the intact alkylated pterin showed the presence of two phosphate residues per pterin. These results established that the pterin isolated from dimethyl sulfoxide reductase contains the phosphoric anhydride of molybdopterin and 5'-GMP, which is designated molybdopterin guanine dinucleotide.

Topics: Chromatography, High Pressure Liquid; Coenzymes; Guanine Nucleotides; Iron-Sulfur Proteins; Metalloproteins; Molybdenum; Molybdenum Cofactors; Oxidoreductases; Pteridines; Pterins; Rhodobacter sphaeroides; Spectrophotometry