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.     

Regulation of Escherichia coli formate hydrogenlyase activity by formate at alkaline pH

Escherichia coli possesses two hydrogenases, Hyd-3 and Hyd-4. These, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-associated formate hydrogenlyases, FHL-1 and FHL-2, both catalyzing the decomposition of formate to H₂ and CO₂ during fermentative growth. FHL-1 is the major pathway at acidic pH whereas FHL-2 is proposed for slightly alkaline pH. In this study, regulation of activity of these pathways by formate has been investigated. In cells grown under fermentative conditions on glucose in the presence of 30 mM formate at pH 7.5, intracellular pH was decreased to 7.1, the activity of Fdh-H raised 3.5-fold, and the production of H₂ became mostly Hyd-3 dependent. These results suggest that at alkaline pH formate increases an activity of Fdh-H and of Hyd-3 both but not of Hyd-4. Received: 27 December 2001 / Accepted: 25 January 2002     

Microbial surface displaying formate dehydrogenase and its application in optical detection of formate

In the present work, NAD⁺-dependent formate dehydrogenase (FDH), encoded by fdh gene from Candida boidinii was successfully displayed on Escherichia coli cell surface using ice nucleation protein (INP) from Pseudomonas borealis DL7 as an anchoring protein. Localization of matlose binding protein (MBP)-INP-FDH fusion protein on the E. coli cell surface was characterized by SDS-PAGE and enzymatic activity assay. FDH activity was monitored through the oxidation of formate catalyzed by cell-surface-displayed FDH with its cofactor NAD⁺, and the production of NADH can be detected spectrometrically at 340 nm. After induction for 24 h in Luria-Bertani medium containing isopropyl-β-d-thiogalactopyranoside, over 80% of MBP-INP-FDH fusion protein present on the surface of E. coli cells. The cell-surface-displayed FDH showed optimal temperature of 50 °C and optimal pH of 9.0. Additionally, the cell-surface-displayed FDH retained its original enzymatic activity after incubation at 4 °C for one month with the half-life of 17 days at 40 °C and 38 h at 50 °C. The FDH activity could be inhibited to different extents by some transition metal ions and anions. Moreover, the E. coli cells expressing FDH showed different tolerance to solvents. The recombinant whole cell exhibited high formate specificity. Finally, the E. coli cell expressing FDH was used to assay formate with a wide linear range of 5–700 μM and a low limit of detection of 2 μM. It is anticipated that the genetically engineered cells may have a broad application in biosensors, biofuels and cofactor regeneration system.     

Kinetic studies of formate dehydrogenase

1. The kinetic mechanism of formate dehydrogenase is a sequential pathway. 2. The binding of the substrates proceeds in an obligatory order, NAD(+) binding first, followed by formate. 3. It seems most likely that the interconversion of the central ternary complex is extremely rapid, and that the rate-limiting step is the formation or possible isomerization of the enzyme-coenzyme complexes. 4. The secondary plots of the inhibitions with HCO(3) (-) and NO(3) (-) are non-linear, which suggests that more than one molecule of each species is able to bind to the same enzyme form. 5. The rate of the reverse reaction with carbon dioxide at pH6.0 is 20 times that with bicarbonate at pH8.0, although no product inhibition could be detected with carbon dioxide. The low rate of the reverse reaction precluded any steady-state analysis as the enzyme concentrations needed to obtain a measurable rate are of the same order as the K(m) values for NAD(+) and NADH.     

Protein engineering of formate dehydrogenase

NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) is one of the best enzymes for the purpose of NADH regeneration in dehydrogenase-based synthesis of optically active compounds. Low operational stability and high production cost of native FDHs limit their application in commercial production of chiral compounds. The review summarizes the results on engineering of bacterial and yeast FDHs aimed at improving their chemical and thermal stability, catalytic activity, switch in coenzyme specificity from NAD+ to NADP+ and overexpression in Escherichia coli cells.     

Dye-linked dehydrogenase activities for formate and formate esters in Amycolatopsis methanolica. Characterization of a molybdoprotein enzyme active with formate esters and aldehydes.

Cell-free extracts of methanol-grown Amycolatopsis methanolica contain dye-linked dehydrogenase activities for formate and methyl formate. Fractionation of the extracts revealed that the (unstable) activity for formate resides in membrane particles, while that for methyl formate belongs to a soluble enzyme that was purified and characterized. The enzyme, indicated as formate-ester dehydrogenase, appeared to be a molybdoprotein (4 Fe, 3 or 4 S, 1 Mo and 1 FAD were found for each enzyme molecule), with a molecular mass of 186 kDa and consisting of two subunits of equal size. Product identification suggests that the formate moiety in the ester becomes hydroxylated to a carbonate group after which the unstable alkyl carbonate decomposes into CO2 and the alcohol moiety. Based on structural and catalytic characteristics, the enzyme appears to be very similar to an enzyme isolated from Comamonas testosteroni [Poels, P. A., Groen, B. W. & Duine, J. A. (1987) Eur. J. Biochem. 166, 575-579] which was at that time considered to be an aldehyde dehydrogenase. Formate-ester dehydrogenase activity appeared to be present in several other bacteria. Possible roles for the A. methanolica enzyme in C1 dissimilation (oxidation of methyl formate to methanol and CO2 or a factor-formate adduct to factor plus CO2) or in general aldehyde oxidation, are discussed.     

A physiological study of formate dehydrogenase,formate oxidase and hydrogenlyase fromEscherichia coli K-12

Escherichia coli was grown under various culture conditions. Variations in the levels of formate dehydrogenase which reacts with methylene blue (MB) or phenazine methosulfate (PMS) (N enzyme), formate dehydrogenase which reacts with benzyl viologen (BV) (H enzyme), formate oxidase and hydrogenlyase were analyzed. It was observed that formate dehydrogenase N and formate oxidase were induced by nitrate and repressed by oxygen. Synthesis of formate dehydrogenase H and hydrogenlyase was induced by formate and repressed by nitrate and oxygen. Selenite was required for the biosynthesis of formate dehydrogenase H and hydrogenlyase. Activity of both formate oxidase and hydrogenlyase was inhibited by azide and KCN but not by N-heptyl hydroxyquinoline-N-oxide (HOQNO); on the other hand, formate oxidase was extremely sensitive to HOQNO. Data were obtained which suggest that cytochromes are not involved in hydrogen formation from formate. Part of this work was carried out when the senior author was visiting Research Biologist in the Laboratory of Dr. J. A. de Mosss at the University of California, San Diego. Thanks are given to Dr. De Moss for his hospitality and advise and to Dr. Warren Butler of the University of California, San Diego for making available his spectrophotometer to carry out cytochrome analyses. Most of this work was sustained by a grant from the Research Corporation, Brown Hazen Fund and the financial help of the C.O.F.A.A. from the Instituto Politécnico Nacional.     

Crystalline formate dehydrogenase from Candida methanolica

Abstract Formate dehydrogenase (EC 1.2.1.2) was purified about 38-fold with an overall yield of 76% from a methanol-utilizing yeast, Candida methanolica (ATCC26175), in 4 steps and, by adding polyethylene glycol, the enzyme was crystallised for the first time. The final preparation appeared to be homogeneous by the criteria of polyacrylamide electrophoresis and analytical centrifugation. Compared with the yeast formate dehydrogenases so far reported, the purified enzyme exhibited higher specific activity (7.52 U/mg).     

Investigation of formate transport through the substrate channel of formate dehydrogenase by steered molecular dynamics simulations

Steered molecular dynamics simulation has revealed the mechanism of formate transport via the substrate channel of formate dehydrogenase. It is shown that the structural organization of the channel promotes the transport of formate anion in spite of the fact that the channel is too narrow even for such a small molecule. The conformational mobility of Arg284 residue, one of the residues forming the wall of the substrate channel, provides for the binding and delivery of formate to the active site.     

The hepatotoxic action of allyl formate

The hepatotoxic action of allyl formate on rat liver has been investigated. Biochemical changes can be detected in the liver cell many hours before the histological changes and it would appear that the toxin has a direct action on the liver parenchymal cell. The results suggest that allyl formate is not the toxic agent but that it is converted via allyl alcohol into acrolein. This reaction requires the presence of alcohol dehydrogenase. Histochemical studies have shown that this enzyme is localized in the periportal region of the liver lobule, and may explain why allyl formate solely produces a periportal necrosis. As glutathione and 1,4-dithiothreitol protect against the early biochemical changes produced by the poison, it is probable that acrolein alkylates proteins and nucleic acids.     

Overexpression of formate dehydrogenase inArabidopsis thaliana resulted in plants tolerant to high concentrations of formate

The potential role played by formate dehydrogenase (FDH) in formate metabolism has been examined by the overexpression of FDH in Arabidopsis thaliana. Three independent transgenic lines were selected and shown to produce elevated amounts of FDH protein with a corresponding elevated FDH activity (2.5-5 fold) over wild-type (WT) plants. Under normal growth conditions, no altered phenotype was observed in these transgenic plants; in growth media supplied with formate, however, significant differences in shoot and root growth, compared to that of WT plants, were observed. WT plants were severely injured if grown in the presence of 16 mmol/L formate, while the transgenic plants were able to grow well. Formate delayed germination of both WT and transgenic seeds at concentrations above 4 mmol/L, but both types of seeds were eventually able to complete more than 95 % germination even at 32 mmol/L formate. Formate markedly inhibited primary root elongation, and its inhibitory action on WT was much stronger than on transgenic plants. Different formate salts affected root elongation similarly, indicating that the formate ion was the major factor inhibiting root growth. Sodium acetate (NaAc), an analogue of formate, also inhibited root elongation, but its action on WT and transgenic plants was the same, indicating that tolerance of transgenic plants to formate toxicity was specific. Transgenic plants showed no significant tolerance to the toxicity of two other one-carbon metabolites, methanol and formaldehyde. A role for FDH in detoxifying formate is proposed.     

Crystal structure of NAD-dependent formate dehydrogenase.

The ternary complex of NAD-dependent formate dehydrogenase (FDH) from the methylotrophic bacterium Pseudomonas sp. 101 (enzyme-NAD-azide) has been crystallised in the space group P2(1)2(1)2(1) with cell dimensions a = 11.60 nm, b = 11.33 nm, c = 6.34 nm. There is 1 dimeric molecule/asymmetric unit. An electron density map was calculated using phases from multiple isomorphous replacement at 0.30 nm resolution. Four heavy atom derivatives were used. The map was improved by solvent flattening and molecular averaging. The atomic model, including 2 x 393 amino acid residues, was refined by the CORELS and PROLSQ packages using data between 1.0 nm and 0.30 nm excluding structure factors less than 1 sigma. The current R factor is 27.1% and the root mean square deviation from ideal bond lengths is 4.2 pm. The FDH subunit is folded into a globular two-domain (coenzyme and catalytic) structure and the active centre and NAD binding site are situated at the domain interface. The beta sheet in the FDH coenzyme binding domain contains an additional beta strand compared to other dehydrogenases. The difference in quaternary structure between FDH and the other dehydrogenases means that FDH constitutes a new subfamily of NAD-dependent dehydrogenases: namely the P-oriented dimer. The FDH nucleotide binding region of the structure is aligned with the three dimensional structures of four other dehydrogenases and the conserved residues are discussed. The amino acid residues which contribute to the active centre and which make contact with NAD have been identified.     

Renewable methanol and formate as microbial feedstocks

1. Download : Download high-res image (86KB)
2. Download : Download full-size image

     

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.     

Improved purification of Candida boidinii formate dehydrogenase

Formate dehydrogenase (FDH, EC 1.2.1.2) from Candida boidinii was purified to homogeneity. The two step procedure comprised anion exchange chromatography (2.9-fold purification, 85% step yield, elution with 35 mM KCl), followed by dye-ligand affinity chromatography on immobilized Cibacron Blue 3GA (1.4-fold purification, 75% step yield, elution with 0.15 mM NAD⁺/2 mM Na₂SO₃). The procedure afforded FDH at 63.8% overall yield and a specific activity of 7.2 units/mg. The purity of the final FDH preparation was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), high performance gel filtration liquid chromatography (gfHPLC) and N-terminal amino acid sequencing. The analytical techniques showed the presence of a single polypeptide chain that corresponds to the molecular weight of 41 kDa (as determined by SDS-PAGE) and 81 kDa (as determined by gfHPLC).