Effect of molybdate and tungstate on the biosynthesis of CO dehydrogenase and the molybdopterin cytosine‐dinucleotide‐type of molybdenum cofactor in Hydrogenophaga pseudoflava

Abstract

The molybdenum-containing iron-sulfur flavoprotein CO dehydrogenase is expressed in a catalytically fully competent form during heterotrophic growth of the aerobic bacterium Hydrogenophaga pseudoflava with pyruvate plus CO. We have adopted these conditions for studying the effect of molybdate (Mo) and tungstate (W) on the biosynthesis of CO dehydrogenase and its molybdopterin (MPT) cytosine-dinucleotide-(MCD)-type molybdenum cofactor. W was taken up by the Mo transport system and, therefore, interfered with Mo transport in an antagonistic way. Depletion of Mo from the growth medium as well as inclusion of excess W both resulted in the absence of intracellular Mo and led to the biosynthesis of CO dehydrogenase species of proper L 2 M 2 S 2 subunit structure that carried the two 2Fe:2S type-I and type-II centers and two FAD molecules. EPR, ultraviolet/visible and CD spectroscopies established the full functionality of the cofactors. Due to the absence of the Mo-MCD cofactor, the enzyme species were catalytically inactive. Unexpectedly, the following cytidine nucleotides were present in inactive CO dehydrogenase: CDP, dCDP, CMP, dCMP, CTP or dCTP. The sum of cytidine nucleotides was two/mol enzyme. The binding specificities of inactive CO dehydrogenase for cytidine nucleotides (oxy Ͼ deoxy; diphosphate Ͼ monophosphate Ͼ triphosphate), and the absence of MPT suggest that, in active CO dehydrogenase, the cytidine diphosphate moiety of Mo-MCD provides the strongest interactions with the protein and determines the specificity for the type of nucleotide. In H. pseudoflava, the biosynthesis of MPT (identified as form A) was independent of Mo. Mo was, however, strictly required for the conversion of MPT to MCD (identified as form-AϪCMP) as well as the insertion of Mo-MCD into CO dehydrogenase. These data support a model for the involvement of Mo in the biosynthesis of the Mo-MCD cofactor and of fully functional CO dehydrogenase in which the synthesis and insertion of Mo-MCD require Mo, and protein synthesis including integration of the FeS-centers and FAD are independent of Mo. Abbreviations. di(cam), di(carboxamidomethyl); MCD, molybdopterin cytosine dinucleotide; MPT, molybdopterin; INT, 1-phenyl-2-(4-iodophenyl)-3-(4-nitrophenyl)-2H-tetrazolium chloride; MPMS, 1-methoxyphenazine methosulfate.Enzyme. Carbon monoxide dehydrogenase (EC 1.2.99.2).nase [3,4], carboxylic acid reductase [5,6] and aldehyde dehydrogenase [7,8]. Most mesophilic, MoϪdependent organisms, when grown in the presence of tungstate, produce either inactive enzymes lacking any metal or W-substituted enzymes that have little or no catalytic activity [2]. This effect of tungstate has been termed in the literature as the antagonistic effect of tungstate over molybdate (e.g. [9]). Pyrococcus furiosus exclusively uses W to synthesize catalytically active forms of the three tungstoenzymes aldehyde ferredoxin oxidoreductase, formaldehyde ferredoxin oxidoreductase and glyceraldehyde-3-phosphate dehydrogenase, and active Mo-containing or V-containing isoenzymes ...

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

“…It has been reported previously that cell extracts from H. pseudoflava grown in the absence of molybdate did not produce MCD (28). In addition, CO dehydrogenase expressed under these conditions was devoid of MCD or MPT, whereas the protein still contained stoichiometric amounts of cytidine moieties, like CDP, dCDP, CMP, dCMP, CTP, or dCTP (28). These observations can now be explained by our results with MocA.…”

“…Another well characterized MCD-containing enzyme is CO dehydrogenase from Hydrogenophaga pseudoflava. It has been reported previously that cell extracts from H. pseudoflava grown in the absence of molybdate did not produce MCD (28). In addition, CO dehydrogenase expressed under these conditions was devoid of MCD or MPT, whereas the protein still contained stoichiometric amounts of cytidine moieties, like CDP, dCDP, CMP, dCMP, CTP, or dCTP (28).…”

Gene Cluster on pAO1 of Arthrobacter nicotinovorans Involved in Degradation of the Plant Alkaloid Nicotine: Cloning, Purification, and Characterization of 2,6-Dihydroxypyridine 3-Hydroxylase

“…The hypothetical MobA protein for the molybdopterin cytidine dinucleotide cofactor is encoded by an ORF which may form a transcriptional unit with ORFs of no known functions but that are presumed to be involved in the assembly of the MoCo holoenzymes, attachment of the enzymes to the cell membrane, and interaction with the respiratory chain (18,28). MobA could deliver, in the context of these proteins, the molybdopterin cytidine dinucleotide cofactor efficiently to the membrane-associated KDH and NDH apoenzymes (16).…”

“…Nicotine, the alkaloid of the tobacco plant, is synthesized as the L-isomer, and the first enzyme to attack L-nicotine is a trimeric, molybdopterin cofactor (MoCo) (most probably in its dinucleotide form [18])-, flavin adenine dinucleotide (FAD)-, and [Fe-S] clustercontaining nicotine dehydrogenase (NDH), which hydroxylates the pyridine ring at position 6 (Fig. …”

Carbon Monoxide Dehydrogenase Activity in Bradyrhizobium japonicum

“…The enzyme is an O 2 -stable, molybdenum-iron-sulfurflavin hydroxylase that catalyzes the oxidation of CO to CO 2 according to the equation CO ϩ H 2 O 3 CO 2 ϩ 2e Ϫ ϩ 2H ϩ . It contains the molybdopterin cytosine dinucleotide-type molybdenum cofactor (29) and [2Fe-2S] centers of type I and type II (2,10). In anaerobic microorganisms, CODHs are nickeliron-sulfur proteins, usually O 2 labile, that function in a variety of energy-yielding pathways.…”