Molecular cloning and sequence analysis of the gene of the molybdenum‐containing aldehyde oxido‐reductase of <i>Desulfovibrio gigas</i>

Thoenes, U.; Flores, Orfeu; Neves, Ana M.; Devreese, Bart; Beeumen, Jozef J. Van; Huber, Robert; Romão, Maria João; LeGall, Jean; Moura, José J. G.; Rodrigues-Pousada, Claudina

doi:10.1111/j.1432-1033.1994.tb18693.x

Cited by 54 publications

(61 citation statements)

References 24 publications

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“…2) and their equivalents in nicotine dehydrogenase of Arthrobacter nicotinovorans (8) exhibited similarities of 74% (32% identity), 83% (42% identity), and 71% (33% identity), respectively. CoxS is 74% similar (41% identical) to aa 1 to 166 of MOP from Desulfovibrio gigas (36). The homology of CoxL to MOP is 64% (28% identity) for aa 22 to 427 and 72% (27% identity) for aa 645 to 780.…”

Section: Coxs Contains Motifs Indicative Of Type I and Ii [2fe-2s]mentioning

confidence: 78%

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

et al. 1995

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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 ...

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Section: Coxs Contains Motifs Indicative Of Type I and Ii [2fe-2s]mentioning

confidence: 78%

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

et al. 1995

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“…They were reported to be under way for the thermophilic archaea (5); the sequence of the P. furiosus aldehyde oxidoreductase gene has been deposited (accession no., X79777). The MOP gene sequence has been published (30).…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of a benzylviologen-linked, tungsten-containing aldehyde oxidoreductase from Desulfovibrio gigas

1995

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Desulfovibrio gigas NCIMB 9332 is a sulfate-reducing bacterium which is able to grow with H 2 , L-and D-lactate, C 4 -dicarboxylic acids, and ethanol as energy sources (17,35). D. gigas not only oxidizes ethanol (to acetate) but it can also store massive amounts of polyglucose (29) which under fermentative conditions can be degraded with ethanol as a major end product (26).Recently, we characterized the NAD-dependent alcohol dehydrogenase from D. gigas and showed it to be an oxygen-labile, decameric enzyme (14). From lactate-grown cells, a molybdenum iron-sulfur protein (MOP) which had 2,6-dichlorophenol-indophenol (DCPIP)-linked aldehyde dehydrogenase activity (1, 31) was purified. In extracts of cells grown on lactate or ethanol, high levels of activity of a coenzyme A-independent, benzylviologen-linked aldehyde dehydrogenase (BV-AlDH) were measured (17). Because at that time only the MOP was known as an enzyme with aldehyde dehydrogenase activity in D. gigas, it was suggested that the BV-AlDH activity was catalyzed by the MOP. Recently, we found that the addition of 0.1 M tungstate to the medium had a strongly stimulatory effect on the growth of D. gigas on ethanol. Omission of tungstate from the medium resulted in a decrease or absence of the BV-AlDH activity and an increase of the DCPIP-linked aldehyde dehydrogenase activity (13). These data and the different characteristics of the enzyme activities in cell extracts strongly suggested that the BV-AlDH and the MOP are two different enzymes.In this report, we describe the purification and characterization of the BV-AlDH and provide evidence that this enzyme belongs to the group of molybdopterin-and tungsten-containing aldehyde:acceptor oxidoreductases. MATERIALS AND METHODSOrganism, cultivation, and preparation of cell extract. D. gigas NCIMB 9332 was cultivated on ethanol, harvested, washed, and stored as described earlier (14), with the following minor modifications. The washing buffer was 50 mM potassium phosphate (pH 7.5) containing 2 mM dithiothreitol (DTT) and 1 mM dithionite. Cell extract was prepared by three successive passages through a French pressure cell operated at 106 MPa and centrifugation (20 min at 48,000 ϫ g and 4ЊC). The cell extract was then used for enzyme purification or stored at Ϫ20ЊC under N 2 until further use.Enzyme assays. Assay systems in cuvettes were made anaerobic by bubbling with N 2 for at least 3 min and were then closed with grey butyl rubber stoppers. The standard assay contained 50 mM potassium phosphate buffer (pH 7.5) and 2 mM benzylviologen in a total volume (after the following additions) of 1 ml. Then, 5 l of a freshly prepared dithionite solution (1.5 mM) and enzyme solution (usually 10 l) were added with microsyringes and the reaction was started by adding acetaldehyde to a final concentration of 1 mM; oxygen was removed from acetaldehyde stock solutions in crimp-seal bottles by replacing the atmosphere with oxygen-free nitrogen twice. Addition of dithionite was necessary to prevent a lag phase in the reaction. Ac...

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“…Cells (Stratagene) and/or E. coli TOP 10FЈ (Invitrogen), prepared according to standard protocols (13), were used to transform the DNA fragments subcloned into the polylinker of the plasmid pZErO TM -1 (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Studies on the Redox Centers of the Terminal Oxidase fromDesulfovibrio gigas and Evidence for Its Interaction with Rubredoxin

Gomes¹,

Silva²,

Oliveira³

et al. 1997

Journal of Biological Chemistry

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128

101

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Rubredoxin-oxygen oxidoreductase (ROO) is the final component of a soluble electron transfer chain that couples NADH oxidation to oxygen consumption in the anaerobic sulfate reducer Desulfovibrio gigas. It is an 86-kDa homodimeric flavohemeprotein containing two FAD molecules, one mesoheme IX, and one Fe-uroporphyrin I per monomer, capable of fully reducing oxygen to water. EPR studies on the native enzyme reveal two components with g values at ϳ2.46, 2.29, and 1.89, which are assigned to low spin hemes and are similar to the EPR features of P-450 hemes, suggesting that ROO hemes have a cysteinyl axial ligation. At pH 7.6, the flavin redox transitions occur at 0 ؎ 15 mV for the quinone/semiquinone couple and at ؊130 ؎ 15 mV for the semiquinone/hydroquinone couple; the hemes reduction potential is ؊350 ؎ 15 mV. Spectroscopic studies provided unequivocal evidence that the flavins are the electron acceptor centers from rubredoxin, and that their reduction proceed through an anionic semiquinone radical. The reaction with oxygen occurs in the flavin moiety. These data are strongly corroborated by the finding that rubredoxin and ROO are located in the same polycistronic unit of D. gigas genome. For the first time, a clear role for a rubredoxin in a sulfate-reducing bacterium is presented.Despite the fact that they are still being considered as strict anaerobes, sulfate reducing bacteria when exposed to oxygen are capable of surviving (1, 2) as well as of taking advantage of its presence in terms of energy conservation (3, 4). In the presence of oxygen, the sulfate reducer Desulfovibrio gigas, uses internal reserves of polyglucose, which is metabolized by the Embden-Meyerhof-Parnas pathway thus generating NADH and ATP (3). In this bacterium a three-component soluble electron transfer chain couples NADH oxidation with oxygen reduction to water, allowing simultaneously for NAD ϩ regeneration and oxygen utilization. The proteins involved are NADH:rubredoxin oxidoreductase (NRO) 1 (5), rubredoxin (Rd) (6), and rubredoxin:oxygen oxidoreductase (ROO) (7), as shown in Scheme I.NRO, the first component of this pathway, contains two subunits of 27 and 32 kDa and is a flavoprotein containing four flavin groups (FMN and FAD) per molecule with reduction potentials (Fl ox /Fl red ) of Ϫ295 and Ϫ325 mV, at pH 7.6. This NADH oxidase reduces D. gigas Rd, a 6-kDa iron protein, with a reduction potential of 0 Ϯ 5 mV. NRO is highly specific toward this redox protein (5). D. gigas desulforedoxin (8), Desulfovibrio desulfuricans Rd (9), or even Desulfovibrio vulgaris Rd (10) are hardly reduced by NRO (5), despite the similarity between the reduction potentials of these proteins. D. gigas Rd was also found to be necessary for the coupling of NRO to the final component of the chain, ROO (7). This enzyme can be seen as a true terminal oxidase since it is capable of catalyzing the four-electron reduction of oxygen to water without the formation of partially reduced oxygen species (7). It is an 86-kDa homodimeric flavohemeprotein, containing tw...

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Molecular cloning and sequence analysis of the gene of the molybdenum‐containing aldehyde oxido‐reductase of Desulfovibrio gigas

Cited by 54 publications

References 24 publications

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

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

Purification and characterization of a benzylviologen-linked, tungsten-containing aldehyde oxidoreductase from Desulfovibrio gigas

Studies on the Redox Centers of the Terminal Oxidase fromDesulfovibrio gigas and Evidence for Its Interaction with Rubredoxin

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