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

NAD(+)-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from methylotrophi c bacterium Pseudomonas sp.101 exhibits the highest stability among the sim ilar type enzymes studied. To obtain further increase in the thermal stabil ity of FDH we used one of general approaches based on hydrophobization of p rotein alpha-helices. Five serine residues in positions 131, 160, 168, 184 and 228 were selected for mutagenesis on the basis of (i) comparative studi es of nine FDH amino acid sequences from different sources and (ii) with th e 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 Se r-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 stabili ty. Mutant S168A was 1.7 times less stable than the wild-type FDH. Double m utants 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 at positive stab ilization effect had the same kinetic parameters as wild-type enzyme. Depen ding on the position of the replaced residue, the single point mutation Ser /Ala increased the FDH stability by 5-24%. Combination of mutations shows n ear additive effect of each mutation to the total FDH stabilization. Four-p oint mutant S(131,160,184,228)A FDH had 1.5 times higher thermal stability compared to the wild-type enzyme, (C) 1999 Federation of European Biochemic al Societies.