flavin-adenine-dinucleotide and molybdopterin-cytosine-dinucleotide

Methanol-grown Amycolatopsis methanolica NCIB 11946 contains a molybdoprotein dehydrogenase, active with aldehydes and formate esters as substrates and with Wurster's blue as electron acceptor, the so-called formate ester dehydrogenase (FEDH) (van Ophem et al., 1992, Eur. J. Biochem. 206, 519-525). It appears now that another molybdoprotein dehydrogenase is present in this organism. This enzyme, indicated here as dye-linked aldehyde dehydrogenase (DL-AlDH), has the same set of cofactors and converts the same type of substrates but with different specificity, and uses 2,6-dichlorophenol-indophenol as sole artificial electron acceptor for those conversions. The enzymes also differ in their quaternary structure, FEDH having an alpha, beta, gamma and DL-AlDH having an alpha, beta, gamma 2 composition. Furthermore, differences exist with respect to the sizes and the N-terminal amino acid sequences of their subunits, indicating that the enzymes derive from different genes. However, neither their substrate specificity nor their induction pattern give a clear indication for distinct physiological roles. Just like other bacterial molybdoprotein dehydrogenases, DL-AlDH consists of three different subunits (87, 35, and 17 kDa) and contains FAD, molybdopterin-cytosine-dinucleotide cofactor, Fe, and acid-labile sulfide in a molar ratio of 1:1:4:4. Although eukaryotic xanthine oxidase and dehydrogenase differ from these prokaryotic dehydrogenases in size and number of their subunits, certain stretches of amino acid sequences show similarity and the magnetic coupling between the Mo and the [2Fe-2S]-1 cluster in DL-AlDH and bovine milk xanthine oxidase is of the same magnitude. In view of this similarity, the topology of the cofactors in the active site of this type of molybdoproteins might be conserved among enzymes from prokaryotic as well as eukaryotic organisms.

Topics: 2,6-Dichloroindophenol; Actinobacteria; Aldehyde Dehydrogenase; Binding Sites; Cytosine Nucleotides; Flavin-Adenine Dinucleotide; Iron; Macromolecular Substances; Protein Conformation; Pterins; Spectrophotometry; Substrate Specificity; Sulfides; Xanthine Oxidase

The CO dehydrogenase structural genes (cox) and orf4 are clustered in the transcriptional order coxM--> coxS--> coxL--> orf4 on the 128-kb megaplasmid pHCG3 of the carboxidotroph Oligotropha carboxidovorans OM5. Sequence analysis suggested association of molybdopterin cytosine dinucleotide and flavin adenine dinucleotide with CoxL and of the [2Fe-2S] clusters with CoxS.

Topics: Aldehyde Oxidoreductases; Amino Acid Sequence; Base Sequence; Binding Sites; Chromosome Mapping; Conserved Sequence; Cytosine Nucleotides; DNA, Bacterial; Flavin-Adenine Dinucleotide; Genes, Bacterial; Iron-Sulfur Proteins; Molecular Sequence Data; Molecular Weight; Molybdenum; Multienzyme Complexes; Multigene Family; Pseudomonas; Pterins; Sequence Homology, Amino Acid

Radioactively labeled carbon monoxide (CO) dehydrogenase has been obtained in good yield and purity from Pseudomonas carboxydoflava grown in the presence of [32P]phosphate. One enzyme molecule contained an average of 8.32 molecules of phosphate. The entire phosphate content was confined to 2 molecules of FAD and 2 molecules of a pterin. These were noncovalently bound. Molybdoenzyme cofactors could be extracted into N-methyl formamide; pterins were isolated by thin-layer chromatography. CO dehydrogenase contained a novel pterin, different from molybdopterin, which was also resolved in other bacterial molybdoenzymes. Therefore, it was tentatively named bactopterin. The characteristic features of bactopterin were as follows. A relative molecular mass, Mr, of 730 which was much greater than that of molybdopterin (330) (Mr values refer to molybdenum-free forms of the cofactors; presumably, the latter were also devoid of the sulfhydryl groups contained in the native compounds). A content of 2 molecules of phosphate/molecule compared to only 1 phosphate in molybdopterin. Bactopterin was three times less susceptible to air oxidation than molybdopterin. Native bactopterin was cleaved by perchloric acid into two phosphorous-containing fragments with Mr of 330 and 420. The smaller one is believed to be very similar to molybdopterin, the larger one was not a pterin but probably contained an aromatic structure.

Topics: Aldehyde Oxidoreductases; Chromatography, Gel; Chromatography, Thin Layer; Coenzymes; Cytosine Nucleotides; Flavin-Adenine Dinucleotide; Formamides; Metalloproteins; Molecular Weight; Molybdenum; Molybdenum Cofactors; Multienzyme Complexes; Perchlorates; Phosphates; Pseudomonas; Pteridines; Pterins