Protein Sci. 2004 Feb;13(2):504-12. Epub 2004 Jan 10.

[DOI Link]

Structural alteration of cofactor specificity in Corynebacterium 2,5-diketo-D-gluconic acid reductase.

Kasha Laboratory, Institute of Molecular Biophysics and Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306-4380, USA.

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.

PMID: 14718658 [PubMed - in process]

This publication is one of the several that describes a structure solved either at the Kasha Laboratory, Institute of Molecular Biophysics or in collaboration with the Institute Faculty. The data used for this structure determination came in full or part from the Macromolecular X-Ray Crystallography Facility.