Bacterial formate dehydrogenase. Increasing the enzyme thermal stability by hydrophobization of alpha-helices

Abstract

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 α-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.