Molecular characterization of the gene cluster coxMSL encoding the molybdenum-containing carbon monoxide dehydrogenase of Oligotropha carboxidovorans

Abstract

The CO dehydrogenase structural genes (cox) and orf4 are clustered in the transcriptional order coxM3 coxS3 coxL3 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.The molybdenum-containing iron-sulfur flavoprotein CODH (26) is the key enzyme in the chemolithoautotrophic utilization of CO by Oligotropha carboxidovorans OM5 (27,29). The large, medium, and small subunits of CODH are encoded by the structural genes coxL, coxM, and coxS, respectively (27). They reside on the 128-kb megaplasmid pHCG3 (20,21). We report here the structural characteristics of the cox genes and the analysis of the deduced amino acid sequences.Abbreviations. The following terms have been abbreviated in this report (abbreviations are shown in parentheses): carbon monoxide dehydrogenase (CODH), aldehyde oxidoreductase (MOP), xanthine dehydrogenase (XDH), flavin adenine dinucleotide (FAD), molybdopterin cytosine dinucleotide (MCD), and amino acid (aa).The strains employed were O. carboxidovorans OM5 (DSM 1227 [29]), Escherichia coli DH5␣ (15), and E. coli K38 (32).The basic recombinant DNA techniques used followed standard protocols (3,25). Plasmids were isolated as described previously (4, 21). The coxS probe (oligonucleotide S) was the 64-fold degenerate 17-mer ATRTGNGCYTTNGCCAT derived from the N-terminal protein sequence MAKAHI of CoxS (12,20). The coxM probe (oligonucleotide M) was the 128-fold degenerate 17-mer ATNCKRTGRTARTCRAA derived from the protein sequence FDYHRI of CoxM (12,20). Deoxyoligonucleotides were 3Ј end labeled with digoxigenin-11-ddUTP. Restriction fragment gene probes were labeled with digoxigenin-11-dUTP by the use of random primers (7). With the 1.45-kb BamHI-EcoRV fragment of pHCG3 (Fig. 1), hybridizations were carried out at 60ЊC and washes were carried out with 1ϫ SSC (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate) (containing 0.1% sodium dodecyl sulfate) at 68ЊC.The 4.86-kb BamHI-HindIII fragment of pHCG3 carrying the structural genes coxMSL was inserted into the vector phagemid pBluescript I KSϩ (Stratagene, Heidelberg, Germany), yielding phagemid pCDH1 (Fig. 1). Sets of nested deletions were introduced bidirectionally into the cloned region by treatment with exonuclease III-S1 nuclease (10) of the Erasea-Base kit (Promega, Madison, Wis.). Sequencing of both strands of cloned DNA with the primers T3 and T7 (Stratagene, La Jolla, Calif.) was performed with the Taq DyeDeoxy Terminator Cycle Sequencing kit in the model 373A DNA sequencing system (Applied Biosystems, Foster City, Calif.). Each nucleotide position was determined with a redundancy of four. The HUSAR program package (Deutsches Krebsforschungszentrum, Heidelberg, Germany) was employed for sequence analysis.DNA sequence and molecular organization of the coxMSL gene cluster. Southern hybridizations with the oligonucleotides M and S identified the 5Ј ...

4-hydroxybenzoyl coenzyme A reductase (dehydroxylating) is required for anaerobic degradation of 4-hydroxybenzoate by Rhodopseudomonas palustris and shares features with molybdenum-containing hydroxylases

“…Insertion of an antibiotic resistance cassette into an open reading frame divergently transcribed from the gene encoding 4-OHBen-CoA ligase, the first enzyme of 4-OHBen degradation, resulted in the generation of a mutant that was unable to grow on 4-OHBen. Sequencing of this region revealed three open reading frames with sequence similarities to bacterial hydroxylating enzymes (21,37,43) as well as to eukaryotic xanthine dehydrogenases (1,28). The work described here suggests that these genes encode the R. palustris 4-OHbenzoyl-CoA reductase (dehydroxylating) and that 4-OHBen is obligatorily metabolized via benzoyl-CoA when it is used as a carbon source for phototrophic growth.…”

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

“…Hydrogenophaga pseudoflava (formerly Pseudomonas carboxydoflava [11)] is a carboxidotrophic bacterium capable of utilizing carbon monoxide (CO) as a source of carbon and energy under aerobic chemolithoautotrophic conditions [12]. CO oxidation is catalyzed by the molybdo iron-sulfur flavoprotein CO dehydrogenase which is a member of the xanthine oxidase family of Mo hydroxylases [13,14]. CO dehydrogenase of H. pseudoflava is a hexamer with an apparent molecular mass of 240 kDa and an L 2 M 2 S 2 subunit structure (Lϭ 70 kDa, M ϭ 33 kDa, Sϭ 17 kDa) [15,16].…”

“…CO oxidation is catalyzed by the molybdo iron-sulfur flavoprotein CO dehydrogenase which is a member of the xanthine oxidase family of Mo hydroxylases [13,14]. CO dehydrogenase of H. pseudoflava is a hexamer with an apparent molecular mass of 240 kDa and an L 2 M 2 S 2 subunit structure (Lϭ 70 kDa, M ϭ 33 kDa, Sϭ 17 kDa) [15,16].…”

The Tungsten Formylmethanofuran Dehydrogenase from Methanobacterium Thermoautotrophicum Contains Sequence Motifs Characteristic for Enzymes Containing Molybdopterin Dinucleotide

“…Thus far only two sequences of molybdopterin-dinucleotidecontaining enzymes are known which do not conform to this sequence fatnily : the nicotine dehydrogenw from Arthrobat:ter rricotinovoruns (Chether-Beck et al, 1994) and the cnrbon monoxide dehydrogcnasc from Oligotrophu carboxidovomns (Schubel et al, 1995). Both enzymes seem to be morc closely related to the molybdopterin-mononucleotide-containing enzymcs, which also form a sequence fatnily, but are not related tn the molybdopteiin-di.nucIec~lide-containing enzyincs (Wvotton el al.. 1991 ;Thoenes et al, 1994).…”