Improvement of the catalytic properties of formate dehydrogenase from
bacterium Staphylococcus aureus by rational design.
NAD +-dependent formate dehydrogenase from the
bacterium Staphylococcus aureus (SauFDH) plays an important role
in the vital activity of these bacteria. The gene encoding SauFDH was
successfully cloned and expressed in our laboratory. Since this enzyme
has the highest k cat value among the described
FDHs and also has a high temperature stability compared to other
proteins, it can be considered as a promising catalyst for regeneration
of NAD(P)H. The main disadvantage of this enzyme are high K
M values. In this work, the principle of rational design
was used to reduce K M. As a result, 9 catalytically
significant positions 119, 194, 196, 217-219, 246, 303, and 323 were
identified, and 16 new mutant forms of SauFDH were obtained and fully
characterized. Substitutions in positions 119 and 194 lead to an
increase of K M NAD+. In the
Ile-Val-Ala-Gly line, position 119 tends to improve NAD
+ binding. K M NAD+
of SauFDH V119G decreased by 27 times compared to the wild-type enzyme.
K M NAD+ Phe194Val decreased by 3.5
times. The catalytic constant for this mutant form practically did not
change. The use of a complex buffer increased the catalytic efficiency 6
times for the mutant with the Phe194Val substitution compared to
wt-SauFDH in a single-component buffer.