Abstract
Corynebacterium
2,5-Diketo-
D
-gluconic acid reductase (2,5-DKGR) catalyzes the reduction of 2,5-diketo-
D
-gluconic acid (2,5-DKG) to 2-Keto-
L
-gulonic acid (2-KLG). 2-KLG is an immediate precursor to
L
-ascorbic acid (vitamin C), and 2,5-DKGR is, therefore, an important enzyme in a novel industrial method for the production of vitamin C. 2,5-DKGR, as with most other members of the aldo-keto reductase (AKR) superfamily, exhibits a preference for NADPH compared to NADH as a cofactor in the stereo-specific reduction of substrate. The application of 2,5-DKGR in the industrial production of vitamin C would be greatly enhanced if NADH could be efficiently utilized as a cofactor. A mutant form of 2,5-DKGR has previously been identified that exhibits two orders of magnitude higher activity with NADH in comparison to the wild-type enzyme, while retaining a high level of activity with NADPH. We report here an X-ray crystal structure of the
holo
form of this mutant in complex with NADH cofactor, as well as thermodynamic stability data. By comparing the results to our previously reported X-ray structure of the
holo
form of wild-type 2,5-DKGR in complex with NADPH, the structural basis of the differential NAD(P)H selectivity of wild-type and mutant 2,5-DKGR enzymes has been identified.