Highly-Active Recombinant Formate Dehydrogenase from Pathogenic Bacterium Staphylococcus aureus: Preparation and Crystallization.

# These authors contributed equally to the work. NAD+-dependent formate dehydrogenase from Staphylococcus aureus (SauFDH) is one of the key enzymes responsible for the survival of this pathogen in the form of biofilms. 3D structure of the enzyme might be helpful in the search for highly specific SauFDH inhibitors that can be used as antibacterial agents exactly against S. aureus biofilms. Here, we prepared a recombinant SauFDH in Escherichia coli cells with a yield of 1 g target protein per liter medium. The developed procedure for the enzyme purification allowed to obtain 400 mg of homogenous enzyme with 61% yield. The specific activity of the purified recombinant SauFDH was 20 U per mg protein, which was 2 times higher than the previously reported activities of formate dehydrogenases. We also found crystallization conditions in the course of two rounds of optimization and obtained 200- and 40-µm crystals for the SauFDH apo- and holoenzymes, respectively. X-ray analysis using synchrotron X-ray sources produced diffraction data sufficient for solving the three-dimensional structures of the apo- and holoenzymes with the resolution of 2.2 and 2.7 Å, respectively. Crystals of the apo- and holoforms of SauFDH had different crystal space groups, which suggest coenzyme binding in the SauFDH holoenzyme.

Anion Binding and Oxidative Modification at the Molybdenum Cofactor of Formate Dehydrogenase from Rhodobacter capsulatus Studied by X-ray Absorption Spectroscopy.

Formate dehydrogenase (FDH) enzymes are versatile catalysts for CO2 conversion. The FDH from Rhodobacter capsulatus contains a molybdenum cofactor with the dithiolene functions of two pyranopterin guanine dinucleotide molecules, a conserved cysteine, and a sulfido group bound at Mo(VI). In this study, we focused on metal oxidation state and coordination changes in response to exposure to O2, inhibitory anions, and redox agents using X-ray absorption spectroscopy (XAS) at the Mo K-edge. Differences in the oxidative modification of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor relative to samples prepared aerobically without inhibitor, such as variations in the relative numbers of sulfido (Mo═S) and oxo (Mo═O) bonds, were observed in the presence of azide (N3-) or cyanate (OCN-). Azide provided best protection against O2, resulting in a quantitatively sulfurated cofactor with a displaced cysteine ligand and optimized formate oxidation activity. Replacement of the cysteine ligand by a formate (HCO2-) ligand at the molybdenum in active enzyme is compatible with our XAS data. Cyanide (CN-) inactivated the enzyme by replacing the sulfido ligand at Mo(VI) with an oxo ligand. Evidence that the sulfido group may become protonated upon molybdenum reduction was obtained. Our results emphasize the role of coordination flexibility at the molybdenum center during inhibitory and catalytic processes of FDH enzymes.

Tailoring of recombinant FDH: effect of histidine tag location on solubility and catalytic properties of Chaetomium thermophilum formate dehydrogenase (CtFDH).

Several protein expression systems can be used to get enzymes in required quantities and study their functions. Incorporating a polyhistidine tag is a beneficial way of getting various enzymes such as FDHs for industrial applications. The NAD+ dependent formate dehydrogenase from Chaetomium thermophilum (CtFDH) can be utilized for interconversion of formate to carbon dioxide coupled with the conversion of NAD+ to NADH. In this study, N-terminal His tagged CtFDH (N-CtFDH) and C-terminal His tagged CtFDH (C-CtFDH) was constructed to learn the effect of His tag location on the activity and kinetic parameters of the enzyme. The solubility of proteins was not affected by tag position, however, an interference on the N-terminal region caused a deterioration in specific activity and the kinetic ability of enzyme. The obtained results indicated that the C-terminus of the enzyme is an appropriate region for tag engineering. The C-CtFDH has an approximately three-fold larger specific activity and two-fold higher catalytic efficiency than N-CtFDH. The results suggest that insertion of a His-tag at the N-terminal or C-terminal end of CtFDH has different effects on the protein and the N-terminal fragment of the protein is crucial for the function of CtFDH.

Spectroscopic and Kinetic Properties of the Molybdenum-containing, NAD+-dependent Formate Dehydrogenase from Ralstonia eutropha.

We have examined the rapid reaction kinetics and spectroscopic properties of the molybdenum-containing, NAD(+)-dependent FdsABG formate dehydrogenase from Ralstonia eutropha. We confirm previous steady-state studies of the enzyme and extend its characterization to a rapid kinetic study of the reductive half-reaction (the reaction of formate with oxidized enzyme). We have also characterized the electron paramagnetic resonance signal of the molybdenum center in its Mo(V) state and demonstrated the direct transfer of the substrate Cα hydrogen to the molybdenum center in the course of the reaction. Varying temperature, microwave power, and level of enzyme reduction, we are able to clearly identify the electron paramagnetic resonance signals for four of the iron/sulfur clusters of the enzyme and find suggestive evidence for two others; we observe a magnetic interaction between the molybdenum center and one of the iron/sulfur centers, permitting assignment of this signal to a specific iron/sulfur cluster in the enzyme. In light of recent advances in our understanding of the structure of the molybdenum center, we propose a reaction mechanism involving direct hydride transfer from formate to a molybdenum-sulfur group of the molybdenum center.

Structural basis for double cofactor specificity in a new formate dehydrogenase from the acidobacterium Granulicella mallensis MP5ACTX8.

Formate dehydrogenases (FDHs) are considered particularly useful enzymes in biocatalysis when the regeneration of the cofactor NAD(P)H is required, that is, in chiral synthesis with dehydrogenases. Their utilization is however limited to the recycling of NAD(+), since all (apart one) of the FDHs characterized so far are strictly specific for this cofactor, and this is a major drawback for their otherwise wide applicability. Despite the many attempts performed to modify cofactor specificity by protein engineering different NAD(+)-dependent FDHs, in the general practice, glucose or phosphite dehydrogenases are chosen for the recycling of NADP(+). We report on the functional and structural characterization of a new FDH, GraFDH, identified by mining the genome of the extremophile prokaryote Granulicella mallensis MP5ACTX8. The new enzyme displays a valuable stability in the presence of many organic cosolvents as well as double cofactor specificity, with NADP(+) preferred over NAD(+) at acidic pH values, at which it also shows the highest stability. The quite low affinities for both cofactors as well as for the substrate formate indicate, however, that the native enzyme requires optimization to be applied as biocatalytic tool. We also determined the crystal structure of GraFDH both as apoprotein and as holoprotein, either in complex with NAD(+) or NADP(+). Noticeably, the latter represents the first structure of an FDH enzyme in complex with NADP(+). This fine picture of the structural determinants involved in cofactor selectivity will possibly boost protein engineering of the new enzyme or other homolog FDHs in view of their biocatalytic exploitation for NADP(+) recycling.

Molybdenum-containing membrane-bound formate dehydrogenase isolated from Citrobacter sp. S-77 having high stability against oxygen, pH, and temperature.

Membrane-bound formate dehydrogenase (FDH) was purified to homogeneity from a facultative anaerobic bacterium Citrobacter sp. S-77. The FDH from Citrobacter sp. S-77 (FDHS77) was a monomer with molecular mass of approximately 150 kDa. On SDS-PAGE, the purified FDHS77 showed as three different protein bands with molecular mass of approximately 95, 87, and 32 kDa, respectively. Based on the N-terminal amino acid sequence analysis, the sequence alignments observed for the 87 kDa protein band were identical to that of the large subunit of 95 kDa, indicating that the purified FDHS77 consisted of two subunits; a 95 kDa large subunit and a 32 kDa small subunit. The purified FDHS77 in this purification did not contain a heme b subunit, but the FDHS77 showed significant activity for formate oxidation, determined by the Vmax of 30.4 U/mg using benzyl viologen as an electron acceptor. The EPR and ICP-MS spectra indicate that the FDHS77 is a molybdenum-containing enzyme, displaying a remarkable O2-stability along with thermostability and pH resistance. This is the first report of the purification and characterization of a FDH from Citrobacter species.

Clostridium acidurici electron-bifurcating formate dehydrogenase.

Cell extracts of uric acid-grown Clostridium acidurici catalyzed the coupled reduction of NAD(+) and ferredoxin with formate at a specific activity of 1.3 U/mg. The enzyme complex catalyzing the electron-bifurcating reaction was purified 130-fold and found to be composed of four subunits encoded by the gene cluster hylCBA-fdhF2.

Clostridium carboxidivorans strain P7T recombinant formate dehydrogenase catalyzes reduction of CO(2) to formate.

Recombinant formate dehydrogenase from the acetogen Clostridium carboxidivorans strain P7(T), expressed in Escherichia coli, shows particular activity towards NADH-dependent carbon dioxide reduction to formate due to the relative binding affinities of the substrates and products. The enzyme retains activity over 2 days at 4°C under oxic conditions.

Characterization of a thermally stable and organic solvent-adaptative NAD+ -dependent formate dehydrogenase from Bacillus sp. F1.

AIMS: To characterize a robust NAD(+) -dependent formate dehydrogenase firstly obtained from a nonmethylotroph, Bacillus sp. F1. METHODS AND RESULTS: The Bacillus sp. F1 NAD(+) -dependent formate dehydrogenase (BacFDH) gene was cloned by TAIL-PCR and heterologous expressed in Escherichia coli. BacFDH was stable at temperatures below 55°C, and the half-life at 60°C was determined as 52·9 min. This enzyme also showed a broad pH stability and retained more than 80% of the activities after incubating in buffers with different pH ranging from 4·5 to 10·5 for 1 h. The activity of BacFDH was significantly enhanced by some metal ions. Moreover, BacFDH exhibited high tolerance to 20% dimethyl sulfoxide, 60% acetone, 10% methanol, 20% ethanol, 60% isopropanol and 20% n-hexane. Like other FDHs, BacFDH displayed strict substrate specificity for formate. CONCLUSION: We isolated a robust formate dehydrogenase, designated as BacFDH, which showed excellent thermal stability, organic solvent stability and a broad pH stability. SIGNIFICANCE AND IMPACT OF THE STUDY: The multi-aspect stability makes BacFDH a competitive candidate for coenzyme regeneration in practical applications of chiral chemicals and pharmaceuticals synthesis with a relatively low cost, especially for the catalysis performed in extreme pH conditions and organic solvents.

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.

Selective oxidation and reduction reactions with cofactor regeneration mediated by galactitol-, lactate-, and formate dehydrogenases immobilized on magnetic nanoparticles.

Rapid immobilization with the one-pot purification of galactitol dehydrogenase (GatDH) and formate dehydrogenase (FDH) is achieved by using iminodiacetic acid (IDA) with chelated Co(2+) modified magnetic nanoparticles as a carrier. Lactate dehydrogenase (LDH) from recombinant Escherichia coli and FDH commencing Candida methylica were used as an auxiliary enzyme for the regeneration of NADH/NAD(+) with a representative synthesis of (S)-1,2-propanediol and l-tagatose starting from hydroxyacetone and galactitol. The affinity magnetic nanoparticles were characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), while the purity of GatDH and FDH was assayed by SDS-PAGE analysis. The immobilized two-enzyme system, reflecting the pH dependence of its constituent enzymes, showed optimal activity at pH 7 and 8 for (S)-1,2-propanediol and l-tagatose production, respectively. The immobilized enzyme system retained up to 70% of its activity after one week of repeated use. The use of affinity magnetic nanoparticles offers the advantage of a one-pot purification of His(6)-tagged GatDH and FDH followed by the production of rare sugar and chiral diol.

Enzymatic reduction of complex redox dyes using NADH-dependent reductase from Bacillus subtilis coupled with cofactor regeneration.

Conventional vat dyeing involves chemical reduction of dyes into their water-soluble leuco form generating considerable amounts of toxic chemicals in effluents. In the present study, a new beta-nicotinamide adenine dinucleotide disodium salt (NADH)-dependent reductase isolated from Bacillus subtilis was used to reduce the redox dyes CI Acid Blue 74, CI Natural Orange 6, and CI Vat Blue 1 into their water-soluble leuco form. Enzymatic reduction was optimized in relation to pH and temperature conditions. The reductase was able to reduce Acid Blue 74 and Natural Orange 6 in the presence of the stoichiometrically consumed cofactor NADH; meanwhile, Vat Blue 1 required the presence of mediator 1,8-dihydroxyanthraquinone. Oxygen from air was used to reoxidize the dyes into their initial forms. The enzymatic reduction of the dyes was studied and the kinetic constants determined, and these were compared to the chemically-reduced leuco form. The enzyme responsible for the reduction showed homology to a NADH-dependent reductase from B. subtilis based on results from the MS/MS peptide mass mapping of the tryptically digested protein. Additionally, the reduction of Acid Blue 74 to its leuco form by reductase from B. subtilis was confirmed using NADH regenerated by the oxidation of formic acid with formate dehydrogenase from Candida boidinii in the same solution.

Formate hydrogenlyase in the hyperthermophilic archaeon, Thermococcus litoralis.

BACKGROUND: Thermococcus litoralis is a heterotrophic facultative sulfur dependent hyperthermophilic Archaeon, which was isolated from a shallow submarine thermal spring. It has been successfully used in a two-stage fermentation system, where various keratinaceous wastes of animal origin were converted to biohydrogen. In this system T. litoralis performed better than its close relative, P. furiosus. Therefore, new alternative enzymes involved in peptide and hydrogen metabolism were assumed in T. litoralis. RESULTS: An about 10.5 kb long genomic region was isolated and sequenced from Thermococcus litoralis. In silico analysis revealed that the region contained a putative operon consisting of eight genes: the fdhAB genes coding for a formate dehydrogenase and the mhyCDEFGH genes encoding a [NiFe] hydrogenase belonging to the group of the H2-evolving, energy-conserving, membrane-bound hydrogenases. Reverse transcription linked quantitative Real-Time PCR and Western blotting experiments showed that the expression of the fdh-mhy operon was up-regulated during fermentative growth on peptides and down-regulated in cells cultivated in the presence of sulfur. Immunoblotting and protein separation experiments performed on cell fractions indicated that the formate dehydrogenase part of the complex is associated to the membrane-bound [NiFe] hydrogenase. CONCLUSION: The formate dehydrogenase together with the membrane-bound [NiFe] hydrogenase formed a formate hydrogenlyase (formate dehydrogenase coupled hydrogenase, FDH-MHY) complex. The expression data suggested that its physiological role is linked to the removal of formate likely generated during anaerobic peptide fermentation.

Protein engineering applications of industrially exploitable enzymes: Geobacillus stearothermophilus LDH and Candida methylica FDH.

Enzymes have become important tools in several industries due to their ability to produce chirally pure and complex molecules with interesting biological properties. The NAD(+)-dependent LDH (lactate dehydrogenase) [bsLDH [Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) LDH] from G. stearothermophilus and the NAD(+)-dependent FDH (formate dehydrogenase) [cmFDH (Candida methylica FDH)] enzyme from C. methylica are particularly crucial enzymes in the pharmaceutical industry and are related to each other in terms of NADH use and regeneration. LDH catalyses the interconversion of pyruvate (oxo acid) and lactate (alpha-hydroxy acid) using the NADH/NAD(+) pair as a redox cofactor. Employing LDH to reduce other oxo acids can generate chirally pure alpha-hydroxy acids of use in the production of pharmaceuticals. One important use of FDH is to regenerate the relatively expensive NADH cofactor that is used by NAD(+)-dependent oxidoreductases such as LDH. Both LDH and FDH from organisms of interest were previously cloned and overproduced. Therefore they are available at a low cost. However, both of these enzymes show disadvantages in the large-scale production of chirally pure compounds. We have applied two routes of protein engineering studies to improve the properties of these two enzymes, namely DNA shuffling and site-directed mutagenesis. Altering the substrate specificity of bsLDH by DNA shuffling and changing the coenzyme specificity of cmFDH by site-directed mutagenesis are the most successful examples of our studies. The present paper will also include the details of these examples together with some other applications of protein engineering regarding these enzymes.

Improving the purification of NAD+-dependent formate dehydrogenase from Candida methylica.

The Candida methylica (cm) recombinant wild type formate dehydrogenase (FDH) gene has been cloned into the pQE-2 TAGZyme expression vector and the 6xHis-tagged FDH gene has been overexpressed in JM105 cells to purify the FDH protein more efficiently, by the use of exopeptidases, TAGZyme Purification System, which has allowed the complete removal of the small N-terminal His-tag. After the purification procedure, 1.2 mg/mL cmFDH protein of >95% purity was obtained. The kinetic parameters of cmFDH have been determined by observing the oxidation of the nicotinamide coenzyme at 340 nm. The results have also been compared to the yield of standard vs. affinity purification of FDH.

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.

Repression of formate dehydrogenase in Solanum tuberosum increases steady-state levels of formate and accelerates the accumulation of proline in response to osmotic stress.

Formate dehydrogenase (FDH, EC 1.2.1.2.) is a soluble mitochondrial enzyme capable of oxidizing formate into CO2 in the presence of NAD+. It is abundant in non-green tissues and scarce in photosynthetic tissues. Under stress, FDH transcripts (and protein) accumulate in leaves, and leaf mitochondria acquire the ability to use formate as a respiratory substrate. In this paper, we describe the analysis of transgenic potato plants under-expressing FDH, obtained in order to understand the physiological function of FDH. Plants expressing low FDH activities were selected and the study was focused on a line (AS23) showing no detectable FDH activity. AS23 plants were morphologically indistinguishable from control plants, and grew normally under standard conditions. However, mitochondria isolated from AS23 tubers could not use formate as a respiratory substrate. Steady-state levels of formate were higher in AS23 leaves and tubers than in control plants. Tubers of untransformed plants oxidized 14C formate into 14CO2 but AS23 tubers accumulated it. In order to reveal a possible phenotype under stress conditions, control and AS23 plants were submitted to drought and cold. These treatments dramatically induced FDH transcripts in control plants but, whatever the growth conditions, no 1.4 kb FDH transcripts were detected in leaves of AS23 plants. Amongst various biochemical and molecular differences between stressed AS23 and control plants, the most striking was a dramatically faster accumulation of proline in the leaves of drought-stressed plants under-expressing FDH.

Purification and properties of the formate dehydrogenase and characterization of the fdhA gene of Sulfurospirillum multivorans.

The soluble periplasmic subunit of the formate dehydrogenase FdhA of the tetrachloroethene-reducing anaerobe Sulfurospirillum multivorans was purified to apparent homogeneity and the gene ( fdhA) was identified and sequenced. The purified enzyme catalyzed the oxidation of formate with oxidized methyl viologen as electron acceptor at a specific activity of 1683 nkat/mg protein. The apparent molecular mass of the native enzyme was determined by gel filtration to be about 100 kDa, which was confirmed by the fdhA nucleotide sequence. fdhA encodes for a pre-protein that differs from the truncated mature protein by an N-terminal 35-amino-acid signal peptide containing a twin arginine motif. The amino acid sequence of FdhA revealed high sequence similarities to the larger subunits of the formate dehydrogenases of Campylobacter jejuni, Wolinella succinogenes, Escherichia coli (FdhN, FdhH, FdhO), and Methanobacterium formicicum. According to the nucleotide sequence, FdhA harbors one Fe(4)/S(4) cluster and a selenocysteine residue as well as conserved amino acids thought to be involved in the binding of a molybdopterin guanidine dinucleotide cofactor.

Purification and characterization of an alpha-haloketone-resistant formate dehydrogenase from Thiobacillus sp. strain KNK65MA, and cloning of the gene.

Thiobacillus sp. strain KNK65MA, which produced an NAD-dependent formate dehydrogenase (FDH) highly resistant to alpha-haloketones, was newly isolated, i.e., the enzyme showed no loss of activity after a 5-h incubation with alpha-haloketones, such as ethyl 4-chloro-3-oxobutanoate. The enzyme was also resistant to SH reagents. The enzyme, purified to homogeneity, was a dimer composed of identical subunits. The specific activity was 7.6 u/mg, and the apparent Km values for formate and NAD+ were 1.6 and 0.048 mM, respectively. The cloned gene of FDH contained one open reading frame (ORF) of 1206 base pairs, predicted to encode a polypeptide of 401 amino acids, with a calculated molecular weight of 44,021; this gene was highly expressed in E. coli cells. The deduced amino acid sequence of this FDH had high identity to other bacterial FDHs.

Purification and characterization of formate dehydrogenase from Ancylobacter aquaticus strain KNK607M, and cloning of the gene.

Ancylobacter aquaticus strain KNK607M, which had high NAD-dependent formate dehydrogenase (FDH) activity, was newly isolated. The enzyme, purified to homogeneity, was a dimer composed of identical subunits with a molecular mass of 44 kDa. The specific activity was 9.5 u/mg, and the enzyme was optimum at pH 6.3 and 50 degrees C, most stable at pH 7.0, and stable at 50 degrees C or lower. The apparent Km values for formate and NAD+ were 2.4 and 0.057 mM, respectively. The enzyme was specific to formate and was inhibited by SH reagents and heavy metal ions. The cloned gene of FDH contained one open reading frame (ORF) of 1206 base pairs, predicted to encode a polypeptide of 401 amino acids, with a calculated molecular weight of 43,895; this gene was highly expressed in E. coli cells. The FDH had high identity to other FDHs, i.e., those of Pseudomonas, Mycobacterium, Moraxella, and Paracoccus, which were 91.3%, 90.8%, 84.2%, and 82.3%, respectively.