The mechanism of formate oxidation by metal-dependent formate dehydrogenases

Metal-dependent formate dehydrogenases (Fdh) from prokaryotic organisms are members of the dimethyl sulfoxide reductase family of mononuclear molybdenum-containing and tungsten-containing enzymes. Fdhs catalyze the oxidation of the formate anion to carbon dioxide in a redox reaction that involves the transfer of two electrons from the substrate to the active site. The active site in the oxidized state comprises a hexacoordinated molybdenum or tungsten ion in a distorted trigonal prismatic geometry. Using this structural model, we calculated the catalytic mechanism of Fdh through density functional theory tools. The simulated mechanism was correlated with the experimental kinetic properties of three different Fdhs isolated from three different Desulfovibrio species. Our studies indicate that the C–H bond break is an event involved in the rate-limiting step of the catalytic cycle. The role in catalysis of conserved amino acid residues involved in metal coordination and near the metal active site is discussed on the basis of experimental and theoretical results.     

The hydrogenases and formate dehydrogenases ofEscherichia coli

Escherichia coli has the capacity to synthesise three distinct formate dehydrogenase isoenzymes and three hydrogenase isoenzymes. All six are multisubunit, membrane-associated proteins that are functional in the anaerobic metabolism of the organism. One of the formate dehydrogenase isoenzymes is also synthesised in aerobic cells. Two of the formate dehydrogenase enzymes and two hydrogenases have a respiratory function while the formate dehydrogenase and hydrogenase associated with the formate hydrogenlyase pathway are not involved in energy conservation. The three formate dehydrogenases are molybdo-selenoproteins while the three hydrogenases are nickel enzymes; all six enzymes have an abundance of iron-sulfur clusters. These metal requirements alone invoke the necessity for a profusion of ancillary enzymes which are involved in the preparation and incorporation of these cofactors. The characterisation of a large number of pleiotropic mutants unable to synthesise either functionally active formate dehydrogenases or hydrogenases has led to the identification of a number of these enzymes. However, it is apparent that there are many more accessory proteins involved in the biosynthesis of these isoenzymes than originally anticipated. The biochemical function of the vast majority of these enzymes is not understood. Nevertheless, through the construction and study of defined mutants, together with sequence comparisons with homologous proteins from other organisms, it has been possible at least to categorise them with regard to a general requirement for the biosynthesis of all three isoenzymes or whether they have a specific function in the assembly of a particular enzyme. The identification of the structural genes encoding the formate dehydrogenase and hydrogenase isoenzymes has enabled a detailed dissection of how their expression is coordinated to the metabolic requirement for their products. Slowly, a picture is emerging of the extremely complex and involved path of events leading to the regulated synthesis, processing and assembly of catalytically active formate dehydrogenase and hydrogenase isoenzymes. This article aims to review the current state of knowledge regarding the biochemistry, genetics, molecular biology and physiology of these enzymes.     

Hydrogenases and formate dehydrogenases of Syntrophobacter fumaroxidans

The syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans possesses two distinct formate dehydrogenases and at least three distinct hydrogenases. All of these reductases are either loosely membrane-associated or soluble proteins and at least one of the hydrogenases is located in the periplasm. These enzymes were expressed on all growth substrates tested, though the levels of each enzyme showed large variations. These findings suggest that both H₂ and formate are involved in the central metabolism of the organism, and that both these compounds may serve as interspecies electron carriers during syntrophic growth on propionate. This revised version was published online in August 2006 with corrections to the Cover Date.     

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

Molybdenum and tungsten are second- and third-row transition elements, respectively, which are found in a mononuclear form in the active site of a diverse group of enzymes that generally catalyze oxygen atom transfer reactions. Mononuclear Mo-containing enzymes have been classified into three families: xanthine oxidase, DMSO reductase, and sulfite oxidase. The proteins of the DMSO reductase family present the widest diversity of properties among its members and our knowledge about this family was greatly broadened by the study of the enzymes nitrate reductase and formate dehydrogenase, obtained from different sources. We discuss in this review the information of the better characterized examples of these two types of Mo enzymes and W enzymes closely related to the members of the DMSO reductase family. We briefly summarize, also, the few cases reported so far for enzymes that can function either with Mo or W at their active site.     

Resolution of distinct selenium-containing formate dehydrogenases from Escherichia coli.

Formate dehydrogenase, a component activity of two alternative electron transport pathways in anaerobic Escherichia coli, has been resolved as two distinguishable enzymes. One, which was induced with nitrate reductase as a component of the formate-nitrate reductase pathway, utilized phenazine methosulfate (PMS) in preference to benzyl viologen (BV) as an artificial electron acceptor and appeared to be exclusively membrane-bound. A second formate dehydrogenase, which was induced as a component of the formate hydrogenlyase pathway, appeared to exist both as a membrane-bound form and as a cytoplasmic enzyme; the cytoplasmic activity was resolved completely from the PMS-linked activity on a sucrose gradient. When E. coli was grown in the presence of 75Se-selenite, a 110,000-dalton selenopeptide, previously shown to be a component of the PMS-linked enzyme, was induced and repressed with this activity. In contrast, an 80,000-dalton selenopeptide was induced and repressed with the BV-linked activity and exhibited a distribution similar to the BV-linked formate dehydrogenase in cell fractions and in sucrose gradients. The results indicate that the two formate dehydrogenases are distinguishable on the basis of their artificial electron acceptor specificity, their cellular localization, and the size of their respective selenoprotein components.     

Chromogenic assessment of the three molybdo-selenoprotein formate dehydrogenases in Escherichia coli

Escherichia coli synthesizes three selenocysteine-dependent formate dehydrogenases (Fdh) that also have a molybdenum cofactor. Fdh-H couples formate oxidation with proton reduction in the formate hydrogenlyase (FHL) complex. The activity of Fdh-H in solution can be measured with artificial redox dyes but, unlike Fdh-O and Fdh-N, it has never been observed by chromogenic activity staining after non-denaturing polyacrylamide gel electrophoresis (PAGE). Here, we demonstrate that Fdh-H activity is present in extracts of cells from stationary phase cultures and forms a single, fast-migrating species. The activity is oxygen labile during electrophoresis explaining why it has not been previously observed as a discreet activity band. The appearance of Fdh-H activity was dependent on an active selenocysteine incorporation system, but was independent of the [NiFe]-hydrogenases (Hyd), 1, 2 or 3. We also identified new active complexes of Fdh-N and Fdh-O during fermentative growth. The findings of this study indicate that Fdh-H does not form a strong complex with other Fdh or Hyd enzymes, which is in line with it being able to deliver electrons to more than one redox-active enzyme complex.     

Molybdenum‐ and tungsten‐containing formate dehydrogenases and formylmethanofuran dehydrogenases: Structure,mechanism, and cofactor insertion

An overview is provided of the molybdenum‐ and tungsten‐containing enzymes that catalyze the interconversion of formate and CO₂, focusing on common structural and mechanistic themes, as well as a consideration of the manner in which the mature Mo‐ or W‐containing cofactor is inserted into apoprotein.     

Classification and enzyme kinetics of formate dehydrogenases for biomanufacturing via CO2 utilization

The reversible interconversion of formate (HCOO^?) and carbon dioxide (CO₂) is catalyzed by formate dehydrogenase (FDH, EC 1.17.1.9). This enzyme can be used as a first step in the utilization of CO₂ as carbon substrate for production of high-in-demand chemicals. However, comparison and categorization of the very diverse group of FDH enzymes has received only limited attention. With specific emphasis on FDH catalyzed CO₂ reduction to HCOO^?, we present a novel classification scheme for FDHs based on protein sequence alignment and gene organization analysis. We show that prokaryotic FDHs can be neatly divided into six meaningful sub-types. These sub-types are discussed in the context of overall structural composition, phylogeny of the gene segment organization, metabolic role, and catalytic properties of the enzymes. Based on the available literature, the influence of electron donor choice on the efficacy of FDH catalyzed CO₂ reduction is quantified and compared. This analysis shows that methyl viologen and hydrogen are several times more potent than NADH as electron donors. Hence, the new FDH classification scheme and the electron donor analysis provide an improved base for developing FDH-facilitated CO₂ reduction as a viable step in the utilization of CO₂ as carbon source for green production of chemicals.     

A sulfurtransferase is essential for activity of formate dehydrogenases in Escherichia coli

l-Cysteine desulfurases provide sulfur to several metabolic pathways in the form of persulfides on specific cysteine residues of an acceptor protein for the eventual incorporation of sulfur into an end product. IscS is one of the three Escherichia coli l-cysteine desulfurases. It interacts with FdhD, a protein essential for the activity of formate dehydrogenases (FDHs), which are iron/molybdenum/selenium-containing enzymes. Here, we address the role played by this interaction in the activity of FDH-H (FdhF) in E. coli. The interaction of IscS with FdhD results in a sulfur transfer between IscS and FdhD in the form of persulfides. Substitution of the strictly conserved residue Cys-121 of FdhD impairs both sulfur transfer from IscS to FdhD and FdhF activity. Furthermore, inactive FdhF produced in the absence of FdhD contains both metal centers, albeit the molybdenum cofactor is at a reduced level. Finally, FdhF activity is sulfur-dependent, as it shows reversible sensitivity to cyanide treatment. Conclusively, FdhD is a sulfurtransferase between IscS and FdhF and is thereby essential to yield FDH activity.     

A comparative study of the thermal stability of formate dehydrogenases from microorganisms and plants

A comparative study of the thermostability of NAD⁺-dependent formate dehydrogenases (FDHs; EC 1.2.1.2) from both methylotrophic bacteria Pseudomonas sp. 101 and Moraxella sp. C1, the methane-utilizing yeast Candida boidinii, and plants Arabidopsis thaliana and Glycine max (soybean) was performed. All the enzymes studied were produced by expression in E. coli cells. The enzymes were irreversibly inactivated in one stage according to first-order reaction kinetics. The FDH from Pseudomonas sp. 101 appeared as the most thermostable enzyme; its counterpart from Glycine max exhibited the lowest stability. The enzymes from Moraxella sp. C1, C. boidinii, and Arabidopsis thaliana showed similar thermostability profiles. The temperature dependence of the inactivation rate constant of A. thaliana FDH was studied. The data of differential scanning calorimetry was complied with the experimental results on the inactivation kinetics of these enzymes. Values of the melting heat were determined for all the enzymes studied.     

Secondary 15N isotope effects on the reactions catalyzed by alcohol and formate dehydrogenases

Secondary 15N isotope effects at the N-1 position of 3-acetylpyridine adenine dinucleotide have been determined, by using the internal competition technique, for horse liver alcohol dehydrogenase (LADH) with cyclohexanol as a substrate and yeast formate dehydrogenase (FDH) with formate as a substrate. On the basis of less precise previous measurements of these 15N isotope effects, the nicotinamide ring of NAD has been suggested to adopt a boat conformation with carbonium ion character at C-4 during hydride transfer [Cook, P. F., Oppenheimer, N. J. & Cleland, W. W. (1981) Biochemistry 20, 1817]. If this mechanism were valid, as N-1 becomes pyramidal an 15N isotope effect of up to 2-3% would be observed. In the present study the equilibrium 15N isotope effect for the reaction catalyzed by LADH was measured as 1.0042 +/- 0.0007. The kinetic 15N isotope effect for LADH catalysis was 0.9989 +/- 0.0006 for cyclohexanol oxidation and 0.997 +/- 0.002 for cyclohexanone reduction. The kinetic 15N isotope effect for FDH catalysis was 1.004 +/- 0.001. These values suggest that a significant 15N kinetic isotope effect is not associated with hydride transfer for LADH and FDH. Thus, in contrast with the deformation mechanism previously postulated, the pyridine ring of the nucleotide apparently remains planar during these dehydrogenase reactions.     

Pilot scale production and isolation of recombinant NAD+- and NADP+-specific formate dehydrogenases.

The expression of the recombinant wild-type NAD+- and mutant NADP+-dependent formate dehydrogenases (EC 1.2.1.2., FDH) from the methanol-utilizing bacterium Pseudomonas sp. 101 in Escherichia coli cells has been improved to produce active and soluble enzyme up to the level of 50% of total soluble proteins. The cultivation process for E. coli/pFDH8a and E. coli/pFDH8aNP cells was optimized and scaled up to a volume of 100 L. A downstream purification process has been developed to produce technical grade NAD+- and NADP+-specific formate dehydrogenases in pilot scale, utilizing extraction in aqueous two-phase systems.     

Effect of interactions between amino acid residues 43 and 61 on thermal stability of bacterial formate dehydrogenases

NAD⁺-dependent formate dehydrogenases (EC 1.2.1.2, FDH) of methylotrophic bacteria Pseudomonas sp. 101 (PseFDH) and Mycobacterium vaccae N10 (MycFDH) exhibit high homology. They differ in two amino acid residues only among a total of 400, i.e., Ile35 and Glu61 in MycFDH substitute for Thr35 and Lys61 as in PseFDH. However, the rate constant for MycFDH thermal inactivation in the temperature range of 54-65°C is 4-6-times higher than the corresponding rate constant for the enzyme from Pseudomonas sp. 101. To clarify the role of these residues in FDH stability the dependence of the apparent rate constant for enzyme inactivation on phosphate concentration was studied. Kinetic and thermodynamic parameters for thermal inactivation were obtained for both recombinant wild-type and mutant forms, i.e., MycFDH Glu61Gln, Glu61Pro, Glu61Lys and PseFDH Lys61Arg. It has been shown that the lower stability of MycFDH compared to that of PseFDH is caused mainly by electrostatic repulsion between Asp43 and Glu61 residues. Replacement of Lys61 with an Arg residue in the PseFDH molecule does not result in an increase in stability.     

Mutagenesis study on the role of a lysine residue highly conserved in formate dehydrogenases and periplasmic nitrate reductases

Lysine 85 (K85) in the primary structure of the catalytic subunit of the periplasmic nitrate reductase (NAP-A) of Ralstonia eutropha H16 is highly conserved in periplasmic nitrate reductases and in the structurally related catalytic subunit of the formate dehydrogenases of various bacterial species. It is located between an [4Fe-4S] center and one of the molybdopterin-guanine dinucleotides mediating the through bonds electron flow to convert the specific substrate of the respective enzymes. To examine the role of K85, the structure of NAP-A of R. eutropha strain H16 was modeled on the basis of the crystal structure from the Desulfovibrio desulfuricans enzyme (Dias et al. Structure Fold Des. 7(1) (1999) 65) and K85 was replaced by site-directed mutagenesis, yielding K85R and K85M, respectively. The specific nitrate reductase activity was determined in periplasmic extracts. The mutant enzyme carrying K85R showed 23% of the wild-type activity, whereas the replacement by a polar, uncharged residue (K85M) resulted in complete loss of the catalytic activity. The reduced nitrate reductase activity of K85R was not due to different quantities of the expressed gene product, as controlled immunologically by NAP-specific antibodies. The results indicate that K85 is optimized for the electron transport flux to reduce nitrate to nitrite in NAP-A, and that the positive charge alone cannot meet further structural requirement for efficient electron flow.     

Alternative NAD+-dependent formate dehydrogenases in the facultative methylotroph Mycobacterium vaccae 10

Mycobacterium vaccae 10 growing in methanol medium synthesizes two inducible alternative NAD(+)-dependent formate dehydrogenases (FDH). In the presence of molybdenum, the dominating form of the enzyme is FDHI with Mr 440 kDa and Km 0.32 mM for sodium formate. FDHI reduced ferricyanide as well as NAD+, and it was reversibly inactivated by formate. NAD+ stabilized FDHI against this inactivation. Under conditions of artificial molybdenum deficiency (tungsten in the medium), the second enzyme (FDHII) appeared with Mr about 93 kDa and Km 8.3 mM for sodium formate, and no FDHI activity was detected. FDHII did not reduce ferricyanide and was not inactivated by formate. The activity of FDHI was restored in tungsten-grown cells by pulse addition of molybdenum under conditions of blocked protein synthesis, suggesting the pre-existence of inactive apo-FDHI.     

Effects of molybdate and tungstate on expression levels and biochemical characteristics of formate dehydrogenases produced by Desulfovibrio alaskensis NCIMB 13491

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 μM molybdate, whereas only the W-FDH was purified from cells cultured in medium supplemented with 10 μM 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.     

Purification of formaldehyde and formate dehydrogenases from pea seeds by affinity chromatography and S-formylglutathione as the intermediate of formaldehyde metabolism

Formaldehyde dehydrogenase (EC 1.2.1.1) and formate dehydrogenase (EC 1.2.1.2) have been isolated in pure form from pea seeds by a rapid procedure which employs column chromatographies on 5′-AMP-Sepharose, Sephacryl S-200, and DE32 cellulose. The apparent molecular weights of formaldehyde and formate dehydrogenases are, respectively, 82,300 and 80,300 by gel chromatography, and they both consist of two similar subunits. The isoelectric point of formaldehyde dehydrogenase is 5.8 and that of formate dehydrogenase is 6.2. The purified formate dehydrogenase gave three corresponding protein and activity bands in electrophoresis and isoelectric focusing on polyacrylamide gel whereas formaldehyde dehydrogenase gave only one band. Formaldehyde dehydrogenase catalyzes the formation of S-formylglutathione from formaldehyde, and glutathione. Formate dehydrogenase can, besides formate, also use S-formylglutathione and two other formate esters as substrates. S-Formylglutathione has a lower K_m value (0.45 mm) than formate (2.1 mm) but the maximum velocity of S-formylglutathione is only 5.5% of that of formate. Pea extracts also contain a highly active S-formylglutathione hydrolase which has been separated from glyoxalase II (EC 3.1.2.6) and partially purified. S-Formylglutathione hydrolase is apparently needed between formaldehyde and formate dehydrogenases in the metabolism of formaldehyde in pea seeds, in contrast to what was recently reported for Hansenula polymorpha, a yeast grown on methanol.     

Two W-containing formate dehydrogenases (CO2-reductases) involved in syntrophic propionate oxidation by Syntrophobacter fumaroxidans.

Two formate dehydrogenases (CO2-reductases) (FDH-1 and FDH-2) were isolated from the syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans. Both enzymes were produced in axenic fumarate-grown cells as well as in cells which were grown syntrophically on propionate with Methanospirillum hungatei as the H2 and formate scavenger. The purified enzymes exhibited extremely high formate-oxidation and CO2-reduction rates, and low Km values for formate. For the enzyme designated FDH-1, a specific formate oxidation rate of 700 U.mg-1 and a Km for formate of 0.04 mm were measured when benzyl viologen was used as an artificial electron acceptor. The enzyme designated FDH-2 oxidized formate with a specific activity of 2700 U.mg-1 and a Km of 0.01 mm for formate with benzyl viologen as electron acceptor. The specific CO2-reduction (to formate) rates measured for FDH-1 and FDH-2, using dithionite-reduced methyl viologen as the electron donor, were 900 U.mg-1 and 89 U.mg-1, respectively. From gel filtration and polyacrylamide gel electrophoresis it was concluded that FDH-1 is composed of three subunits (89 +/- 3, 56 +/- 2 and 19 +/- 1 kDa) and has a native molecular mass of approximately 350 kDa. FDH-2 appeared to be a heterodimer composed of a 92 +/- 3 kDa and a 33 +/- 2 kDa subunit. Both enzymes contained tungsten and selenium, while molybdenum was not detected. EPR spectroscopy suggested that FDH-1 contains at least four [2Fe-2S] clusters per molecule and additionally paramagnetically coupled [4Fe-4S] clusters. FDH-2 contains at least two [4Fe-4S] clusters per molecule. As both enzymes are produced under all growth conditions tested, but with differences in levels, expression may depend on unknown parameters.