Department of Chemical Enzymology, Faculty of Chemistry, Lomonosov
Moscow State University, Moscow, 119992, Russia.
From analysis of Ramachandran plot for NAD+-dependent formate
dehydrogenase from the methylotrophic bacterium Pseudomonas sp. 101 (FDH,
EC 1.2.1.2), five amino acid residues with non-optimal values phi and
psi have been located in beta- and pi-turns of the FDH polypeptide
chain, e.g., Asn136, Ala191, Tyr144, Asn234, and His263. To clarify
their role in the enzyme stability, the residues were replaced with Gly
by means of site-directed mutagenesis. The His263Gly mutation caused FDH
destabilization and a 1.3-fold increase in the monomolecular
inactivation rate constant. The replacements Ala191Gly and Asn234Gly had
no significant effect on the stability. The mutations Asn136Gly and
Tyr144Gly resulted in higher thermal stability and decreased the
inactivation rate by 1.2- and 1.4-fold, respectively. The stabilizing
effect of the Tyr144Gly mutation was shown to be additive when
introduced into the previously obtained mutant FDH with enhanced thermal
stability.
