guanosine-monophosphate and molybdenum-cofactor

The molybdenum cofactor is an important cofactor, and its biosynthesis is essential for many organisms, including humans. Its basic form comprises a single molybdopterin (MPT) unit, which binds a molybdenum ion bearing three oxygen ligands via a dithiolene function, thus forming Mo-MPT. In bacteria, this form is modified to form the bis-MPT guanine dinucleotide cofactor with two MPT units coordinated at one molybdenum atom, which additionally contains GMPs bound to the terminal phosphate group of the MPTs (bis-MGD). The MobA protein catalyzes the nucleotide addition to MPT, but the mechanism of the biosynthesis of the bis-MGD cofactor has remained enigmatic. We have established an in vitro system for studying bis-MGD assembly using purified compounds. Quantification of the MPT/molybdenum and molybdenum/phosphorus ratios, time-dependent assays for MPT and MGD detection, and determination of the numbers and lengths of Mo-S and Mo-O bonds by X-ray absorption spectroscopy enabled identification of a novel bis-Mo-MPT intermediate on MobA prior to nucleotide attachment. The addition of Mg-GTP to MobA loaded with bis-Mo-MPT resulted in formation and release of the final bis-MGD product. This cofactor was fully functional and reconstituted the catalytic activity of apo-TMAO reductase (TorA). We propose a reaction sequence for bis-MGD formation, which involves 1) the formation of bis-Mo-MPT, 2) the addition of two GMP units to form bis-MGD on MobA, and 3) the release and transfer of the mature cofactor to the target protein TorA, in a reaction that is supported by the specific chaperone TorD, resulting in an active molybdoenzyme.

Topics: Coenzymes; Escherichia coli; Escherichia coli Proteins; Guanosine Monophosphate; Humans; Metalloproteins; Molecular Chaperones; Molybdenum; Molybdenum Cofactors; Oxidation-Reduction; Oxidoreductases, N-Demethylating; Phosphates; Protein Binding; Pteridines; Sulfurtransferases; X-Ray Absorption Spectroscopy

All mononuclear molybdoenzymes bind molybdenum in a complex with an organic cofactor termed molybdopterin (MPT). In many bacteria, including Escherichia coli, molybdopterin can be further modified by attachment of a GMP group to the terminal phosphate of molybdopterin to form molybdopterin guanine dinucleotide (MGD). This modification reaction is required for the functioning of many bacterial molybdoenzymes, including the nitrate reductases, dimethylsulfoxide (DMSO) and trimethylamine-N-oxide (TMAO) reductases, and formate dehydrogenases. The GMP attachment step is catalyzed by the cellular enzyme MobA.. The crystal structure of the 21.6 kDa E. coli MobA has been determined by MAD phasing with selenomethionine-substituted protein and subsequently refined at 1. 35 A resolution against native data. The structure consists of a central, predominantly parallel beta sheet sandwiched between two layers of alpha helices and resembles the dinucleotide binding Rossmann fold. One face of the molecule bears a wide depression that is lined by a number of strictly conserved residues, and this feature suggests that this is where substrate binding and catalysis take place.. Through comparisons with a number of structural homologs, we have assigned plausible functions to several of the residues that line the substrate binding pocket. The enzymatic mechanism probably proceeds via a nucleophilic attack by MPT on the GMP donor, most likely GTP, to produce MGD and pyrophosphate. By analogy with related enzymes, this process is likely to require magnesium ions.

Topics: Amino Acid Sequence; Bacterial Proteins; Binding Sites; Catalytic Domain; Coenzymes; Consensus Sequence; Crystallography, X-Ray; Escherichia coli; Escherichia coli Proteins; Evolution, Molecular; Guanosine Monophosphate; Metalloproteins; Models, Molecular; Molecular Sequence Data; Molybdenum Cofactors; Protein Binding; Protein Conformation; Protein Structure, Secondary; Pteridines; Recombinant Fusion Proteins; Selenomethionine; Sequence Alignment; Sequence Homology, Amino Acid

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 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