NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) widely occurs in nature. FDH consists of two identical subunits and contains neither prosthetic groups nor metal ions. This type of FDH was found in different microorganisms (including pathogenic ones), such as bacteria, yeasts, fungi, and plants. As opposed to microbiological FDHs functioning in cytoplasm, plant FDHs localize in mitochondria. Formate dehydrogenase activity was first discovered as early as in 1921 in plant; however, until the past decade FDHs from plants had been considerably less studied than the enzymes from microorganisms. This review summarizes the recent results on studying the physiological role, properties, structure, and protein engineering of plant formate dehydrogenases.

It has been shown by an X-ray structural analysis that the amino acid residues Ala198, which are located in the coenzyme-binding domain of NAD+-dependent formate dehydrogenases (EC 1.2.1.2., FDH) from bacteria Pseudomonas sp.101 and Moraxella sp. C-1 (PseFDH and MorFDH, respectively), have non-optimal values of the angles ψ and φ. These residues were replaced with Gly by site-directed mutagenesis. The mutants PseFDH A198G and MorFDH A198G were expressed in E.coli cells and obtained in active and soluble forms with more than 95% purity. The study of thermal inactivation kinetics showed that the mutation A198G results in a 2.5- fold increase in stability compared to one for the wild-type enzymes. Kinetic experiments indicate that A198G replacement reduces the KMNAD+ value from 60 to 35 and from 80 to 45 μM for PseFDH and MorFDH, respectively, while the KMHCOO- value remains practically unchanged. Amino acid replacement A198G was also added to the mutant PseFDH D221S with the coenzyme specificity changed from NAD+ to NADP+. In this case, an increase in thermal stability was also observed, but the influence of the mutation on the kinetic parameters was opposite: KM increased from 190 to 280 μM and from 43 to 89 mM for NADP+ and formate, respectively. According to the data obtained, inference could be drawn that earlier formate dehydrogenase from bacterium Pseudomonas sp. 101 was specific to NADP+, but not to NAD+.

Penicillin acylases (PA) are widely used for the production of semi-synthetic β-lactam antibiotics and chiral compounds. In this review, the latest achievements in the production of recombinant enzymes are discussed, as well as the results of PA type G protein engineering.

The analysis of the 3D model structure of the ternary complex of recombinant formate dehydrogenase from soya Glycine max (EC 1.2.1.2., SoyFDH) with bound NAD+ and an inhibitor azide ion revealed the presence of hydrophobic Phe290 in the coenzyme-binding domain. This residue should shield the enzyme active site from solvent. On the basis of the alignment of plant FDHs sequences, Asp, Asn and Ser were selected as candidates to substitute Phe290. Computer modeling indicated the formation of two (Ser and Asn) or three (Asp) new hydrogen bonds in such mutants. The mutant SoyFDHs were expressed in Escherichia coli, purified and characterized. All amino acid substitutions increased K(м)(HCOO-) from 1.5 to 4.1-5.0 mM, whereas the K(м)(NAD+) values remained almost unchanged in the range from 9.1 to 14.0 μM, which is close to wt-SoyFDH (13.3 μM). The catalytic constants for F290N, F290D and F290S mutants of SoyFDH equaled 2.8, 5.1 and 4.1 s⁻¹, respectively; while that of the wild-type enzyme was 2.9 s⁻¹. The thermal stability of all mutant SoyFDHs was much higher compared with the wild-type enzyme. The differential scanning calorimetry data were in agreement with the results of thermal inactivation kinetics. The mutations F290S, F290N and F290D introduced into SoyFDH increased the T(m) values by 2.9°C, 4.3°C and 7.8°C, respectively. The best mutant F290D exhibited thermal stability similar to that of FDH from the plant Arabidopsis thaliana and exceeded that of the enzymes from the yeast Candida boidinii and the bacterium Moraxella sp. C1.

Kinetic studies on hydrogen peroxide–induced inactivation of mutant formate dehydrogenase from Pseudomonas sp. 101 (PseFDH Cys255Ala) suggest a simple bimolecular mechanism for enzyme reaction with the inactivation agent. In the excess of hydrogen peroxide, the decrease in enzyme activity follows first–order kinetics. Therefore, the first–order effective inactivation kinetic constants determined for various FDH forms at a constant H2O2 concentration can be used as a quantitative measure of the enzyme stability. It was shown that two cysteine residues located in the active site formate– and coenzyme–binding domains (Cys145 and Cys255, respectively) make similar contributions to the enzyme stability, while the contribution of Cys354 is insignificant. The inactivation kinetics of wild–type PseFDH, mutant PseFDH Cys145Ser/Cys255Ala, and FDH produced under stress conditions by bacterium Staphylococcus aureus, higher plants Arabidopsis thaliana, and soya Glycine max, was studied. It was found that the stress–induced FDHs are at least 20 times more stable than the nonstress–induced PseFDH from Pseudomonas sp. 101 grown on methanol.

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Recombinant formate dehydrogenase (FDH, EC 1.2.1.2) from soy Glycine max (SoyFDH) has the lowest values of Michaelis constants for formate and NAD+ among all studied formate dehydrogenases from different sources. Nevertheless, it also has the lower thermal stability compared to enzymes from bacteria and yeasts. The alignment of full sequences of FDHs from different sources as well as structure of apo- and holo-forms of SoyFDH has been analyzed. Ten mutant forms of SoyFDH were obtained by site-directed mutagenesis. All of them were purified to homogeneity and their thermal stability and substrate specificity were studied. Thermal stability was investigated by studying the inactivation kinetics at different temperatures and by differential scanning calorimetry (DSC). As a result, single-point (Ala267Met) and double mutants (Ala267Met/Ile272Val) were found to be more stable than the wild-type enzyme at high temperatures. The stabilization effect depends on temperature, and at 52°C it was 3.6- and 11-fold, respectively. These mutants also showed higher melting temperatures in DSC experiments - the differences in maxima of the melting curves (T(m)) for the single and double mutants were 2.7 and 4.6°C, respectively. For mutations Leu24Asp and Val127Arg, the thermal stability at 52°C decreased 5- and 2.5-fold, respectively, and the T(m) decreased by 3.5 and 1.7°C, respectively. There were no differences in thermal stability of six mutant forms of SoyFDH - Gly18Ala, Lys23Thr, Lys109Pro, Asn247Glu, Val281Ile, and Ser354Pro. Analysis of kinetic data showed that for the enzymes with mutations Val127Arg and Ala267Met the catalytic efficiency increased 1.7- and 2.3-fold, respectively.

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