Alteration of coenzyme specificity in halophilic NAD(P)+ glucose dehydrogenase by site-directed mutagenesis

Structural analysis of glucose dehydrogenase from Haloferax mediterranei revealed that the adenosine 2′-phosphate of NADP+ was stabilized by the side chains of Arg207 and Arg208. To investigate the structural determinants for coenzyme specificity, several mutants involving residues Gly206, Arg207 and Arg208 were engineered and kinetically characterized. The single mutants G206D and R207I were less efficient with NADP+ than the wild type, and the double and triple mutants G206D/R207I and G206D/R207I/R208N showed no activity with NADP+.In the single mutant G206D, the relation kcat/KNAD+kcat/KNAD+ was 1.6 times higher than in the wild type, resulting in an enzyme that preferred NAD+ over NADP+. The single mutation was sufficient to modify coenzyme specificity, whereas other dehydrogenases usually required more than one or two mutations to change coenzyme specificity. However, the highest reaction rates were reached with the double mutant G206D/R207I and with coenzyme NAD+, where the kcat was 1.6 times higher than the kcat of the wild-type enzyme with NADP+. However, catalytic efficiency with NAD+ was lower, as the Km value for coenzyme was 77 times higher than the wild type with NADP+.