Redesign of the coenzyme specificity in L-lactate dehydrogenase from Bacillus stearothermophilus using site-directed mutagenesis and media engineering

L-Lactate dehydrogenase (LDH) from Bacillus stearothermophilus is a redox e nzyme which has a strong preference for NADH over NADPH as coenzyme, To exc lude NADPH from the coenzyme-binding pocket, LDH contains a conserved aspar tate residue at position 52, However, this residue is probably not solely r esponsible for the NADH specificity. In this report we examine the possibil ities of altering the coenzyme specificity of LDH by introducing a range of different point mutations in the coenzyme-binding domain. Furthermore, aft er choosing the mutant with the highest selectivity for NADPH, we also inve stigated the possibility of further altering the coenzyme specificity by ad ding an organic solvent to the reaction mixture. The LDH mutant, I51K:D52S, exhibited a 56-fold increased specificity to NADPH over the wild-type LDH in a reaction mixture containing 15% methanol. Furthermore, the NADPH turno ver number of this mutant was increased almost fourfold as compared with wi ld-type LDH, To explain the altered coenzyme specificity exhibited by the D 52SI51K double mutant, molecular dynamics simulations were performed.