Effects of tungstate on the growth of Desulfovibrio gigas NCIMB 9332 and other sulfate-reducing bacteria with ethanol as a substrate

Hensgens, Charles M.H.; Nienhuis-Kuiper, Manny E.; Hansen, T. A.

doi:10.1007/s002030050116

Cited by 12 publications

(25 citation statements)

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“…Several of the tungsten-containing enzymes show aldehyde dehydrogenase activity (2,22,30,31,41,44). Yet for acetylene hydratase of P. acetylenicus, no such activity could be demonstrated.…”

Section: Discussionmentioning

confidence: 99%

“…Formylmethanofuran dehydrogenases of M. wolfei and M. thermoautotrophicum, aldehyde ferredoxin oxidoreductase of P. furiosus, and formaldehyde oxidoreductase of T. litoralis can be purified only under strict exclusion of oxygen, because they lose activity within a few minutes to several hours under air (6,31,38). Sensitivity to oxygen has been described recently also for the benzyl viologen-dependent aldehyde dehydrogenase of D. gigas, which is supposed to contain tungsten (22).…”

Section: Discussionmentioning

confidence: 99%

“…However, a tungsten-containing formate dehydrogenase has been purified also from the mesophilic bacterium C. formicoaceticum (13). There is evidence for tungsten-containing enzymes in other mesophilic anaerobic bacteria as well, e.g., in Desulfovibrio gigas and other sulfatereducing bacteria (22), and even in aerobic bacteria (21,26).…”

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Purification and characterization of acetylene hydratase of Pelobacter acetylenicus, a tungsten iron-sulfur protein

1995

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Acetylene hydratase of the mesophilic fermenting bacterium Pelobacter acetylenicus catalyzes the hydration of acetylene to acetaldehyde. Growth of P. acetylenicus with acetylene and specific acetylene hydratase activity depended on tungstate or, to a lower degree, molybdate supply in the medium. The specific enzyme activity in cell extract was highest after growth in the presence of tungstate. Enzyme activity was stable even after prolonged storage of the cell extract or of the purified protein under air. However, enzyme activity could be measured only in the presence of a strong reducing agent such as titanium(III) citrate or dithionite. The enzyme was purified 240-fold by ammonium sulfate precipitation, anion-exchange chromatography, size exclusion chromatography, and a second anion-exchange chromatography step, with a yield of 36%. The protein was a monomer with an apparent molecular mass of 73 kDa, as determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The isoelectric point was at pH 4.2. Per mol of enzyme, 4.8 mol of iron, 3.9 mol of acid-labile sulfur, and 0.4 mol of tungsten, but no molybdenum, were detected. The K m for acetylene as assayed in a coupled photometric test with yeast alcohol dehydrogenase and NADH was 14 M, and the V max was 69 mol ⅐ min ؊1 ⅐ mg of protein ؊1. The optimum temperature for activity was 50؇C, and the apparent pH optimum was 6.0 to 6.5. The N-terminal amino acid sequence gave no indication of resemblance to any enzyme protein described so far.The strictly anaerobic fermenting bacterium Pelobacter acetylenicus converts the unsaturated hydrocarbon ethine (trivial name, acetylene) to acetate and ethanol via acetaldehyde as an intermediate (36). The first step of the fermentation pathway, the hydration of acetylene to acetaldehyde, is catalyzed by the enzyme acetylene hydratase. The reaction is highly exergonic: C 2 H 2 ϩ H 2 O 3 CH 3 CHO (⌬GЊЈ ϭ Ϫ111.9 kJ/mol) (36). Until recently, attempts to demonstrate this enzyme activity in cell extracts of P. acetylenicus have failed (27,36). Also the aerobic acetylene-degrading bacteria Mycobacterium lacticola, Nocardia rhodochrous, Rhodococcus strain A1, and Rhodococcus rhodochrous have been reported to convert acetylene to acetaldehyde (7,16,20,25); the reaction was proposed to be catalyzed by an acetylene hydratase activity. Yet acetylene hydratase activity could be demonstrated in cell extracts of Rhodococcus strain A1 only when the assay was performed under anoxic conditions (16). Therefore, acetylene appears to be the only hydrocarbon that is converted in the presence and absence of molecular oxygen by the same type of enzyme (36).Tungsten-containing enzymes have been reported so far to catalyze redox reactions of a low reduction potential (E 0 Ј Ͻ Ϫ400 mV), such as those involving formate dehydrogenase of Clostridium thermoaceticum (51) and Clostridium formicoaceticum (13), aldehyde ferredoxin oxidoreductase of Pyrococcus furiosus (30), formaldehyde ferredoxin oxidoreductase of Thermococcus litoralis (31), carboni...

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“…Several of the tungsten-containing enzymes show aldehyde dehydrogenase activity (2,22,30,31,41,44). Yet for acetylene hydratase of P. acetylenicus, no such activity could be demonstrated.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Purification and characterization of acetylene hydratase of Pelobacter acetylenicus, a tungsten iron-sulfur protein

1995

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“…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.…”

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confidence: 84%

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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“…So far they have been obtained from methanogenic, acetogenic, and fermentative anaerobes, all but one of which (Clostridium formicoaceticum) are thermophilic or hyperthermophilic. Very recent evidence indicates that tungstoenzymes are also present in mesophilic sulfate-reducing bacteria (18) and in some aerobic methylotrophs (16). Purified tungstoenzymes include (i) formate dehydrogenase from Clostridium thermoaceticum (50,53) and C. formicoaceticum (12), (ii) carboxylic acid reductase (CAR) from the same two organisms (49,52), (iii) aldehyde ferredoxin oxidoreductase (AOR) from Pyrococcus furiosus (32), (iv) formaldehyde ferredoxin oxidoreductase (FOR) from Thermococcus litoralis and P. furiosus (19,33), (v) formylmethanofuran dehydrogenase from Methanobacterium wolfei (43) and Methanobacterium thermoautotrophicum (6), and (vi) glyceraldehyde-3-phosphate (GAP) ferredoxin oxidoreductase (GAPOR) from P. furiosus (34).…”

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Purification, characterization, and metabolic function of tungsten-containing aldehyde ferredoxin oxidoreductase from the hyperthermophilic and proteolytic archaeon Thermococcus strain ES-1

1995

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Thermococcus strain ES-1 is a strictly anaerobic, hyperthermophilic archaeon that grows at temperatures up to 91؇C by the fermentation of peptides. It is obligately dependent upon elemental sulfur (S 0 ) for growth, which it reduces to H 2 S. Cell extracts contain high aldehyde oxidation activity with viologen dyes as electron acceptors. The enzyme responsible, which we term aldehyde ferredoxin oxidoreductase (AOR), has been purified to electrophoretic homogeneity. AOR is a homodimeric protein with a subunit M r of approximately 67,000. It contains molybdopterin and one W, four to five Fe, one Mg, and two P atoms per subunit. ) for its AOR. The most efficient substrates for Thermococcus strain ES-1 AOR were the aldehyde derivatives of transaminated amino acids. This suggests that the enzyme functions to oxidize aldehydes generated during amino acid catabolism, although the possibility that AOR generates aldehydes from organic acids produced by fermentation cannot be ruled out.Although molybdenum-containing enzymes are ubiquitous in nature (45), a biological requirement for tungsten (W), an analog of molybdenum (Mo), has only recently been established (4). Indeed, the first naturally occurring W-containing enzyme was purified only in the early 1980s (53). Interest in tungstoenzymes has greatly intensified in the last few years, and several different types have been purified (3). So far they have been obtained from methanogenic, acetogenic, and fermentative anaerobes, all but one of which (Clostridium formicoaceticum) are thermophilic or hyperthermophilic. Very recent evidence indicates that tungstoenzymes are also present in mesophilic sulfate-reducing bacteria (18) and in some aerobic methylotrophs (16). Purified tungstoenzymes include (i) formate dehydrogenase from Clostridium thermoaceticum (50, 53) and C. formicoaceticum (12), (ii) carboxylic acid reductase (CAR) from the same two organisms (49, 52), (iii) aldehyde ferredoxin oxidoreductase (AOR) from Pyrococcus furiosus (32), (iv) formaldehyde ferredoxin oxidoreductase (FOR) from Thermococcus litoralis and P. furiosus (19,33), (v) formylmethanofuran dehydrogenase from Methanobacterium wolfei (43) and Methanobacterium thermoautotrophicum (6), and (vi) glyceraldehyde-3-phosphate (GAP) ferredoxin oxidoreductase (GAPOR) from P. furiosus (34). Amino-terminal amino acid sequence analyses (23, 32) indicate strong homology between CAR, FOR, and AOR, suggesting that these enzymes, all of which can utilize aldehydes as substrates, are closely related. On the other hand, formylmethanofuran dehydrogenase and GAPOR (34) show little or no N-terminal homology to the aldehyde-oxidizing enzymes or to each other (data have not been reported for formate dehydrogenase).The best characterized of the tungstoenzymes at the molecular level is AOR from the hyperthermophile P. furiosus (3,31,32). The gene for AOR has been cloned and sequenced (23), and its crystal structure has been determined to a resolution of 2.3 Å (0.23 nm) (10). These data show that the enzyme is a homodimer wit...

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Effects of tungstate on the growth of Desulfovibrio gigas NCIMB 9332 and other sulfate-reducing bacteria with ethanol as a substrate

Cited by 12 publications

References 0 publications

Purification and characterization of acetylene hydratase of Pelobacter acetylenicus, a tungsten iron-sulfur protein

Purification and characterization of acetylene hydratase of Pelobacter acetylenicus, a tungsten iron-sulfur protein

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

Purification, characterization, and metabolic function of tungsten-containing aldehyde ferredoxin oxidoreductase from the hyperthermophilic and proteolytic archaeon Thermococcus strain ES-1

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