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

Abstract

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...

Mo and W bis-MGD enzymes: nitrate reductases and formate dehydrogenases

“…Different authors seem to agree that Wcontaining isoenzymes represent, apparently, less evolved forms or ancestors of the Mo-containing enzymes [3,5,51]. In this context, the existing Wcontaining enzymes, such as D. gigas and S. fumaroxidans Fdhs [3,4], would represent a group of enzymes that cannot adopt Mo to accomplish a specific function in living organisms, which along evolution were substituting Mo for W in enzymes.…”

“…The inverse reaction, the conversion of CO 2 to formate, has been demonstrated in a few cases, and this activity was found to be exclusive for W-Fdh proteins [4]. Consequently, it was suggested that the reduction of CO 2 to formate is W-dependent, whereas the inverse reaction in anaerobes is performed exclusively by Mo-containing enzymes [4]. Additional studies should be performed to verify whether the mechanism proposed for E. coli Fdh-H is valid for all formate dehydrogenases [13] and if the conversion of CO 2 to formate is exclusive for W-Fdh proteins [5].…”

“…Mo-containing enzymes are found in almost all forms of life [1], whereas W-containing enzymes seem to be exclusive for organisms such as hyperthermofilic archaea that live in extreme environments [2]. However, W-containing enzymes have been found in organisms that do not need extreme conditions [3,4,5], suggesting a more important role for tungsten [6]. Some of the mononuclear W-containing enzymes described present active sites similar to the Mo enzymes, but they have different 3D structures and have been classified in a different group [2,7].…”

“…1) might be a sulfur atom in D. gigas Fdh, in which case the reaction mechanism should be re-evaluated. The inverse reaction, the conversion of CO 2 to formate, has been demonstrated in a few cases, and this activity was found to be exclusive for W-Fdh proteins [4]. Consequently, it was suggested that the reduction of CO 2 to formate is W-dependent, whereas the inverse reaction in anaerobes is performed exclusively by Mo-containing enzymes [4].…”

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

“…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].…”