V. I. Tishkov

Catalytic mechanism and application of formate dehydrogenase

NAD+-dependent formate dehydrogenase (FDH) is an abundant enzyme that plays an important role in energysupply of methylotrophic microorganisms and in response to stress in plants. FDH belongs to the superfamily of D-specific 2-hydroxy acid dehydrogenases. FDH is widely accepted as a model enzyme to study the mechanism of hydride ion transfer in the active center of dehydrogenases because the reaction catalyzed by the enzyme is devoid of proton transfer steps and implies a substrate with relatively simple structure. FDH is also widely used in enzymatic syntheses of optically active compounds as a versatile biocatalyst for NAD(P)H regeneration consumed in the main reaction. This review covers the late developments in cloning genes of FDH from various sources, studies of its catalytic mechanism and physiological role, and its application for new chiral syntheses.

D-Amino acid oxidase: structure, catalytic mechanism, and practical application.

D-Amino acid oxidase (DAAO) is a FAD-dependent enzyme that plays an important role in microbial metabolism, utilization of endogenous D-amino acids, regulation of the nervous system, and aging in mammals. DAAO from yeasts Rhodotorula gracilis and Trigonopsis variabilis are used to convert cephalosporin C into 7-aminocephalosporanic acid, the precursor of other semi-synthetic cephalosporins. This review summarizes the recent data on the enzyme localization, physiological role, gene cloning and expression, and the studies on the enzyme structure, stability, catalytic mechanism, and practical applications.

A novel, efficient regenerating method of NADPH using a new formate dehydrogenase

Abstract A NADPH regenerating system using a new, protein engineered formate dehydrogenase (FDH) is investigated. The new enzyme is the first known NAD(P)H dependent FDH. It can be successfully employed in synthesis with other enzymes requiring a NADPH regeneration. All advantages of the known NAD(H) dependent FDHs in these enzyme coupled synthesis can now be transferred to NADP(H) dependent systems.

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.

Engineering of coenzyme specificity of formate dehydrogenase from Saccharomyces cerevisiae.

A eukaryotic formate dehydrogenase (EC 1.2.1.2, FDH) with its substrate specificity changed from NAD(+) to NADP(+) has been constructed by introducing two single-point mutations, Asp(196)-->Ala (D196A) and Tyr(197)-->Arg (Y197R). The mutagenesis was based on the results of homology modelling of a NAD(+)-specific FDH from Saccharomyces cerevisiae (SceFDH) using the Pseudomonas sp.101 FDH (PseFDH) crystal structure as a template. The resulting model structure suggested that Asp(196) and Tyr(197) mediate the absolute coenzyme specificity of SceFDH for NAD(+).

Synthesis of chiral ε-lactones in a two-enzyme system of cyclohexanone mono-oxygenase and formate dehydrogenase with integrated bubble-free aeration

Abstract A two-enzyme system consisting of a cyclohexanone mono-oxygenase from Acinetobacter NCIMB 9871 and a protein-engineered formate dehydrogenase for the regeneration of the cofactor NADPH was used for the synthesis of chiral e-lactones. 4-Methylcyclohexanone was used as the model substrate yielding ( S )-(−)-5-methyl-oxepane-2-one with high chemical and enantiomeric purity. Syntheses were carried out in a repetitive-batch reactor with integrated bubble-free aerationby means of a thin-walled silicon tube.

Cloning of formate dehydrogenase gene from a methanol-utilizing bacterium Mycobacterium vaccae N10.

The gene of NAD+-dependent formate dehydrogenase (FDH) from Mycobacterium vaccae N10 was cloned into Escherichia coli by hybridization with digoxigenin-labeled DNA probes, which were prepared by amplification of the chromosomal DNA from the bacterium by the polymerase chain reaction with degenerate primers. The primers were designed on the basis of the most conserved parts of known sequences of FDH from different organisms. An open-reading frame of 1200 bp exhibited extremely high sequence similarity to the FDH gene of Pseudomonas sp. 101. The deduced amino acid sequence of FDH from Mycobacterium vaccae N10 (McFDH) was identical to that of Pseudomonas sp. 101 (PsFDH) except for two amino acid residues: isoleucine-35 (threonine in PsFDH) and glutamate-61 (lysine in PsFDH). The physicochemical properties of both enzymes appeared to be closely similar to each other, but the thermostability of McFDH was a little lower than that of PsFDH. To examine the role of the two amino acid residues in the thermostability of the enzymes, glutamate-61 of McFDH was replaced by glutaminyl, prolyl and lysyl residues by site-directed mutagenesis. All the mutant enzymes showed higher thermostability than the wild-type McFDH. The negative charge of glutamate-61 contributes to the stability of the wild-type enzyme being lower than that of PsFDH.

Structure‐guided alteration of coenzyme specificity of formate dehydrogenase by saturation mutagenesis to enable efficient utilization of NADP+

Formate dehydrogenase from Candida boidinii (CboFDH) catalyses the oxidation of formate anion to carbon dioxide with concomitant reduction of NAD+ to NADH. CboFDH is highly specific to NAD+ and virtually fails to catalyze the reaction with NADP+. Based on structural information for CboFDH, the loop region between β‐sheet 7 and α‐helix 10 in the dinucleotide‐binding fold was predicted as a principal determinant of coenzyme specificity. Sequence alignment with other formate dehydrogenases revealed two residues (Asp195 and Tyr196) that could account for the observed coenzyme specificity. Positions 195 and 196 were subjected to two rounds of site‐saturation mutagenesis and screening and enabled the identification of a double mutant Asp195Gln/Tyr196His, which showed a more than 2 × 107‐fold improvement in overall catalytic efficiency with NADP+ and a more than 900‐fold decrease in the efficiency with NAD+ as cofactors. The results demonstrate that the combined polar interactions and steric factors comprise the main structural determinants responsible for coenzyme specificity. The double mutant Asp195Gln/Tyr196His was tested for practical applicability in a cofactor recycling system composed of cytochrome P450 monooxygenase from Bacillus subtilis, (CYP102A2), NADP+, formic acid and ω‐(p‐nitrophenyl)dodecanoic acid (12‐pNCA). Using a 1250‐fold excess of 12‐pNCA over NADP+ the first order rate constant was determined to be equal to kobs = 0.059 ± 0.004 min−1.

D-Amino acid oxidase: Physiological role and applications

D-Amino acids play a key role in regulation of many processes in living cells. FAD-dependent D-amino acid oxidase (DAAO) is one of the most important enzymes responsible for maintenance proper level of D-amino acids. The most interesting and important data for regulation of the nervous system, hormone secretion, and other processes by D-amino acids as well as development of different diseases under changed DAAO activity are presented. The mechanism of regulation is complex and multi-parametric because the same enzyme simultaneously influences the level of different D-amino acids, which can result in opposing effects. Use of DAAO for diagnostic and therapeutic purposes is also considered.

SITE-DIRECTED MUTAGENESIS OF THE FORMATE DEHYDROGENASE ACTIVE CENTRE : ROLE OF THE HIS332-GLN313 PAIR IN ENZYME CATALYSIS

Gln313 and His332 residues in the active centre of NAD+‐dependent formate dehydrogenase (EC 1.2.1.2, FDH) from the bacterium Pseudomonas sp. 101 are conserved in all FDHs and are equivalent to the glutamate‐histidine pair in active sites of d‐specific 2‐hydroxyacid dehydrogenases. Two mutants of formate dehydrogenase from Pseudomonas sp. 101, Gln313Glu and His332Phe, have been obtained and characterised. The Gln313Glu mutation shifts the pK of the group controlling formate binding from less than 5.5 in wild‐type enzyme to 7.6 thus indicating that Gln313 is essential for the broad pH affinity profile towards substrate. His332Phe mutation leads to a complete loss of enzyme activity. The His332Phe mutant is still able to bind coenzyme but not substrate or analogues. The role of histidine in the active centre of FDH is discussed. The protonation state of His332 is not critical for catalysis but vital for substrate binding. A partial positive charge on the histidine imidazole, required for substrate binding, is provided via tight H‐bond to the Gln313 carboxamide.