Molybdenum Incorporation in Tungsten Aldehyde Oxidoreductase Enzymes from
            <i>Pyrococcus furiosus</i>

Sevcenco, Ana–Maria; Bevers, Loes E.; Pinkse, Martijn W. H.; Krijger, Gerard C.; Wolterbeek, Hubert Th.; Verhaert, Peter; Hagen, Wilfred R.; Hagedoorn, Peter‐Leon

doi:10.1128/jb.00270-10

Cited by 41 publications

(38 citation statements)

References 34 publications

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“…We could not identify either of the riboswitch sequences close to the genes for D. vulgaris FDHs. So, at this stage, it is difficult to speculate on the mechanism of regulation that can discriminate between Mo and W. The recent report of low Mo incorporation in the P. furiosus tungsten AORs, when the organism is grown with very low W and high Mo concentrations, reveals that also in this organism a selective intracellular mechanism is present to discriminate between the two metals, even when high intracellular concentrations of Mo are present in this tungsten-dependent organism (43).…”

Section: Discussionmentioning

confidence: 99%

“…Substitution of molybdenum for tungsten has been reported for an acetylene hydratase, in which the Mo-substituted enzyme shows 60% activity of the natural tungsten protein (6). The Pyrococcus furiosus AOR tungstoenzymes were recently shown to be able to incorporate Mo, albeit with no activity (43). Given the high similarity of the two elements, it is interesting to understand how biological systems have developed solutions to discriminate between the two.…”

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confidence: 99%

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Tungsten and Molybdenum Regulation of Formate Dehydrogenase Expression in Desulfovibrio vulgaris Hildenborough

et al. 2011

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Formate is an important energy substrate for sulfate-reducing bacteria in natural environments, and both molybdenum-and tungsten-containing formate dehydrogenases have been reported in these organisms. In this work, we studied the effect of both metals on the levels of the three formate dehydrogenases encoded in the genome of Desulfovibrio vulgaris Hildenborough, with lactate, formate, or hydrogen as electron donors. Using Western blot analysis, quantitative real-time PCR, activity-stained gels, and protein purification, we show that a metal-dependent regulatory mechanism is present, resulting in the dimeric FdhAB protein being the main enzyme present in cells grown in the presence of tungsten and the trimeric FdhABC 3 protein being the main enzyme in cells grown in the presence of molybdenum. The putatively membrane-associated formate dehydrogenase is detected only at low levels after growth with tungsten. Purification of the three enzymes and metal analysis shows that FdhABC 3 specifically incorporates Mo, whereas FdhAB can incorporate both metals. The FdhAB enzyme has a much higher catalytic efficiency than the other two. Since sulfate reducers are likely to experience high sulfide concentrations that may result in low Mo bioavailability, the ability to use W is likely to constitute a selective advantage.Formate is a key metabolite in anaerobic habitats, arising as a metabolic product of bacterial fermentations and functioning as a growth substrate for many microorganisms (for example, methanogens and sulfate-reducing bacteria [SRB]). Formate is also an intermediate in the energy metabolism of several prokaryotes and a crucial compound in many syntrophic associations, whereby organisms live close to the thermodynamic limit (30,45). Recent reports indicate that formate plays an even more important role in anaerobic microbial metabolism than previously considered (14,24,27). The key enzyme in formate metabolism is formate dehydrogenase (FDH) (50), a member of the dimethyl sulfoxide (DMSO) reductase family. It catalyzes the reversible two-electron oxidation of formate or reduction of CO 2 and plays a role in energy metabolism and carbon fixation. In anaerobic microorganisms, FDH includes a molybdenum or tungsten bis-(pyranopterin guanidine dinucleotide) cofactor and iron-sulfur clusters (20, 41) and shows great variability in quaternary structure, physiological redox partner, and cellular location (7,23,38,50).FDH was the first enzyme shown to naturally incorporate tungsten, at a time when this element was considered to be mostly an antagonist to molybdenum (2, 52). Since then, several tungstoenzymes have been isolated and characterized, mainly but not exclusively from archaeal organisms, including FDHs, formylmethanofuran dehydrogenases (FMDH), aldehyde oxidoreductases (AOR) (not belonging to the xanthine oxidase family), and acetylene hydratase (3,4,20,25,31,41). FDHs and FMDHs can naturally incorporate either tungsten or molybdenum. Since these two elements have very similar chemical and catalytic properties,...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Tungsten and Molybdenum Regulation of Formate Dehydrogenase Expression in Desulfovibrio vulgaris Hildenborough

et al. 2011

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“…Despite the fact that these metal atoms share similar chemical properties, it is still unknown why highly homologous enzymes that catalyze the same reaction and that are isolated from closely related organisms incorporate either Mo or W in the active site (1,5,9,36,42).…”

Section: Discussionmentioning

confidence: 99%

Effects of Molybdate and Tungstate on Expression Levels and Biochemical Characteristics of Formate Dehydrogenases Produced by Desulfovibrio alaskensis NCIMB 13491

et al. 2011

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Formate dehydrogenases (FDHs) are enzymes that catalyze the formate oxidation to carbon dioxide and that contain either Mo or W in a mononuclear form in the active site. In the present work, the influence of Mo and W salts on the production of FDH by Desulfovibrio alaskensis NCIMB 13491 was studied. Two different FDHs, one containing W (W-FDH) and a second incorporating either Mo or W (Mo/W-FDH), were purified. Both enzymes were isolated from cells grown in a medium supplemented with 1 M molybdate, whereas only the W-FDH was purified from cells cultured in medium supplemented with 10 M tungstate. We demonstrated that the genes encoding the Mo/W-FDH are strongly downregulated by W and slightly upregulated by Mo. Metal effects on the expression level of the genes encoding the W-FDH were less significant. Furthermore, the expression levels of the genes encoding proteins involved in molybdate and tungstate transport are downregulated under the experimental conditions evaluated in this work. The molecular and biochemical properties of these enzymes and the selective incorporation of either Mo or W are discussed.Sulfate-reducing bacteria (SRB) are anaerobic prokaryotes that have a major economic impact in biocorrosion processes of ferrous metals and water pollution (18,21,22). These organisms use electrons donors, such as hydrogen, organic acids, or alcohols, to reduce sulfate to sulfide (43).Desulfovibrio species are sulfate-reducing bacteria in which the oxidation of formate to carbon dioxide is catalyzed by Moor W-containing formate dehydrogenases (FDHs). This reaction provides electrons that are transferred probably through the periplasmic cytochrome network to the cytoplasm to be used for sulfate reduction (19). The formate dehydrogenases identified in Desulfovibrio species belong to the dimethyl sulfoxide (DMSO) reductase family and have been isolated as heterodimeric (1, 6) or heterotrimeric (9, 41) proteins. The ␣ subunit of these enzymes (ϳ90 kDa) contains the active site and one [4Fe-4S] cluster. The active site is constituted by a Mo or a W atom coordinated by two pyranopterin molecules, a selenocysteine (typical in this type of FDH), and a sulfur atom. The ␤ subunit (ϳ29 kDa) contains three additional [4Fe-4S] clusters, which together with the heme groups harbored in the third subunit (ϳ16 kDa) of some FDHs form part of the enzyme electron transfer pathway (7,20,26,37 . Note that the D. alaskensis G20 genome corresponds to that deposited as D. desulfuricans G20. The reclassification of D. desulfuricans G20 as D. alaskensis G20 has been proposed but not yet officially adopted (29). Amino acid sequence alignment of the proteins encoded by these genes shows a high identity percentage (48 to 75%) with the homologous proteins isolated and characterized from other Desulfovibrio species. Likewise, D. alaskensis G20 FDHs appear as heterodimeric soluble enzymes composed of ␣ and ␤ subunits. Three major features are found in the amino acid sequence of the ␣ subunit: a twin-arginine motif [RRXF(L/I)K] characteristic...

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“…This finding suggests that the main function of the chaperone is to keep the protein unfolded for a longer time period and therefore extend the time frame for cofactor assembly and metal insertion during biosynthesis of AH. To get more information about this issue, experiments with radioactive metal isotopes are planned (29).…”

Section: Discussionmentioning

confidence: 99%

Exploring the Active Site of the Tungsten, Iron-Sulfur Enzyme Acetylene Hydratase

2011

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The soluble tungsten, iron-sulfur enzyme acetylene hydratase (AH) from mesophilic Pelobacter acetylenicus is a member of the dimethyl sulfoxide (DMSO) reductase family. It stands out from its class as it catalyzes a nonredox reaction, the addition of H 2 O to acetylene (HOC'COH) to form acetaldehyde (CH 3 CHO). Caught in its active W(IV) state, the high-resolution three-dimensional structure of AH offers an excellent starting point to tackle its unique chemistry and to identify catalytic amino acid residues within the active site cavity: Asp13 close to W(IV) coordinated to two molybdopterin-guanosine-dinucleotide ligands, Lys48 which couples the [4Fe-4S] cluster to the W site, and Ile142 as part of a hydrophobic ring at the end of the substrate access channel designed to accommodate the substrate acetylene. A protocol was developed to express AH in Escherichia coli and to produce active-site variants which were characterized with regard to activity and occupancy of the tungsten and iron-sulfur centers. By this means, fusion of the N-terminal chaperone binding site of the E. coli nitrate reductase NarG to the AH gene improved the yield and activity of AH and its variants significantly. Results from site-directed mutagenesis of three key residues, Asp13, Lys48, and Ile142, document their important role in catalysis of this unusual tungsten enzyme.Molybdenum and tungsten are the only transition metals of the second (Mo) and third (W) row of the periodic table of elements with known biological functions (7). In their biologically active form, both metals are bound to the cofactor molybdopterin (Moco), which is present in all molybdenum and tungsten enzymes with the exception of nitrogenase, where molybdenum is coordinated to a large iron-sulfur cluster, MoFe 7 S 9 (9). Virtually all organisms including plants and mammals use either molybdenum or tungsten proteins in important metabolic pathways (35

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Molybdenum Incorporation in Tungsten Aldehyde Oxidoreductase Enzymes from Pyrococcus furiosus

Cited by 41 publications

References 34 publications

Tungsten and Molybdenum Regulation of Formate Dehydrogenase Expression in Desulfovibrio vulgaris Hildenborough

Tungsten and Molybdenum Regulation of Formate Dehydrogenase Expression in Desulfovibrio vulgaris Hildenborough

Effects of Molybdate and Tungstate on Expression Levels and Biochemical Characteristics of Formate Dehydrogenases Produced by Desulfovibrio alaskensis NCIMB 13491

Exploring the Active Site of the Tungsten, Iron-Sulfur Enzyme Acetylene Hydratase

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