Alteration of the formate dehydrogenase isoelectric point by rational design

In order to extend the pH stability optimum for NAD⁺-dependent formate dehydrogenase (FDH, EC 1.2.1.2) from the bacterium Pseudomonas sp. 101 (PseFDH), four mutant enzymes with Lys112Pro, Lys231Ala, Lys231Ser, and Lys317Asn substitutions were obtained by site-directed mutagenesis. The choice of the mutation sites and the types of substituting amino acids were based on the alignment of amino acid sequences of FDHs from various sources and the analysis of the three-dimensional structure of PseFDH. The kinetic properties and temperature stability were studied for all obtained mutant forms. It is shown that the substitutions in positions 112 and 231 slightly improved the kinetic properties; meanwhile, the Lys317Asn mutant possessed a decreased affinity for the coenzyme. A thermal stability assay for the obtained mutants revealed that the substitutions in positions 112 and 231 result in just a slight destabilization of the enzyme, while Lys317Asn substitution causes a significant decrease in thermal stability. The isoelectric point was decreased by 0.1 points for all obtained mutant forms.