Cofactor specificity in the aldo-keto reductase (AKR) superfamily has been well studied, and several groups have reported the rational alteration of cofactor specificity in these enzymes Although most efforts have focused on mesostable AKRs, several putative AKRs have recently been identified from hyperthermophiles The few that have been characterized exhibit a strong preference for NAD(H) as a cofactor, in contrast to the NADP(H) preference of the mesophilic AKRs Using the design rules elucidated from mesostable AKRs, we introduced two site directed mutations in the cofactor binding pocket to investigate cofactor specificity in a thermostable AKR, AdhD, which is an alcohol dehydrogenase from Pyrococcus furiosus The resulting double mutant exhibited significantly improved activity and broadened cofactor specificity as compared to the wild-type Results of previous pre-steady-state kinetic experiments suggest that the high affinity of the mesostable AKRs for NADP(H) stems from a conformational change upon cofactor binding which is mediated by interactions between a canonical irginine and the 2'-phosphate of the cofactor Pre steady state kinetics with AdhD and the new mutants show a rich conformational behavior that is independent of the canonical irginine or the 2' phosphate Additionally, experiments with the highly active double mutant using NADPH as a cofactor demonstrate an unprecedented transient behavior when the binding mechanism appears to be dependent on cofactor concentration These results suggest that the structural features involved in cofactor specificity in the AKRs are conserved within the superfamily but the dynamic interactions of the enzyme with cofactors ire unexpectedly complex Biotechnol Bioeng 2010,107 763-774 (C) 2010 Wiley Periodicals, Inc