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(+).
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.
NAD + -dependent formate dehydrogenase (EC 1.2.1.2, FDH) from methylotrophic bacterium Pseudomonas sp.101 exhibits the highest stability among the similar type enzymes studied. To obtain further increase in the thermal stability of FDH we used one of general approaches based on hydrophobization of protein K K-helices. Five serine residues in positions 131, 160, 168, 184 and 228 were selected for mutagenesis on the basis of (i) comparative studies of nine FDH amino acid sequences from different sources and (ii) with the analysis of the ternary structure of the enzyme from Pseudomonas sp.101. Residues Ser-131 and Ser-160 were replaced by Ala, Val and Leu. Residues Ser-168, Ser-184 and Ser-228 were changed into Ala. Only Ser/Ala mutations in positions 131, 160, 184 and 228 resulted in an increase of the FDH stability. Mutant S168A was 1.7 times less stable than the wild-type FDH. Double mutants S(131,160)A and S(184,228)A and the four-point mutant S(131,160,184,228)A were also prepared and studied. All FDH mutants with a positive stabilization effect had the same kinetic parameters as wild-type enzyme. Depending on the position of the replaced residue, the single point mutation Ser/Ala increased the FDH stability by 5^24%. Combination of mutations shows near additive effect of each mutation to the total FDH stabilization. Four-point mutant S(131,160,184,228)A FDH had 1.5 times higher thermal stability compared to the wild-type enzyme.z 1999 Federation of European Biochemical Societies.
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+.
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