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NAD(P)+
-dependent formate dehydrogenase (FDH, EC 1.2.1.2.) catalyzes the oxidation of formate ion with the coupled reduction of NAD(P)+
to
NAD(P)H. Previously, in our laboratory, a genetic construct was obtained with
the soyfdh2 gene encoding isoenzyme 2 of formate dehydrogenase from soybean
Glycine max (SoyFDH). In this construct the nucleotide sequence encoding the
signal peptide responsible for the transport of the pro-enzyme into the mitochondria of plant cells (the SoyFDH_L enzyme) was deleted. In this work, a second variant of SoyFDH_S was obtained, in which, compared to SoyFDH_L, the sequence
at the N-terminus was reduced and changed to mimic the N-terminus sequence in
FDH from Pseudomonas sp.101 bacteria. Next, a sequence of six histidine residues
(His-tag) was added to the N-terminus of the long and short forms of SoyFDH.
All four SoyFDH variants were expressed in E. coli BL21(DE3)CodonPlus, these
enzymes were purified, their kinetic parameters were determined, and thermal
stability was studied. In the case of SoyFDH_L, which is similar to the natural
variant of the enzyme, both with and without His-tag, the expression level is two
times higher compared to the truncated variant. The addition of His-tag to the
N-terminus of enzymes reduces the level of expression. Changing the sequence
of the N-terminus, as well as introducing the His-tag sequence to the N-terminus,
does not significantly affect thermal stability of the enzymes at temperatures of
50–56 °C. However, due to the higher values of the activation enthalpy ΔH≠
of the
thermal inactivation process, the shortened form at normal temperatures is 3 times
more stable than the natural one. A comparison of the kinetic parameters of the two SoyFDH variants shows that the catalytic constants are the same, but the long
version of SoyFDH_L has lower values KM
HCOO–
, and the short version has lower
KM
NAD+
values. The introduction of His-tag into the N-terminus of enzymes does not
affect their kinetic parameters.

NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) from methylotrophic bacterium Pseudomonas sp.101 (PseFDH) has one of the highest thermal stability among all known enzymes of this group. The introduction of a number of amino acid substitutions into PseFDH made it possible to obtain a multipoint mutant PseFDH SM4S enzyme with even higher temperature and chemical stability. Previously, we showed that the introduction of additional single point replacements S131A, or S160A, or E170D into PseFDH SM4S led to further stabilization of the enzyme. In this work, based on the PseFDH SM4S S131A mutant, new mutant FDHs obtained, in which, compared to PseFDH SM4S, we added double S131A/E170D (M2), triple S131A/S160A/E170D (M3) and quadruple S131A/S160A/ E170D/S145A (PseFDH SM4A M3) amino acids replacements. The new PseFDH mutants were overexpressed in E. coli cells, puri ed and characterized. The S131A/E170D and S131A/S160A/E170D changes provided further improving thermal stability. The introduction of the S145A substitution into PseFDH SM4S M4 leads to a signi cant decrease in KMNAD+ and KMHCOO- while maintaining the catalytic constant at the same level. This mutant form can be successfully used in NADH regeneration systems, as well as for the detection of NAD+ and formate in biological systems.