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Structural alteration of cofactor specificity in Corynebacterium 2,5‐diketo‐D‐gluconic acid reductase
Author(s) -
Sanli Gulsah,
Banta Scott,
Anderson Stephen,
Blaber Michael
Publication year - 2004
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.03450704
Subject(s) - cofactor , nad+ kinase , reductase , gluconic acid , chemistry , stereochemistry , enzyme , corynebacterium glutamicum , ascorbic acid , biochemistry , mutant , biosynthesis , food science , gene
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.