Structural alteration of cofactor specificity in Corynebacterium 2,5-diketo-D-gluconic acid reductase.
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.
Sanli
Gulsah
G
Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4380, USA.
Banta
Scott
S
Anderson
Stephen
S
Blaber
Michael
M
D002942:Circular Dichroism; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism*; D003352:Corynebacterium / Q000201:enzymology*; D018360:Crystallography, X-Ray; D005942:Gluconates / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D003067:Coenzymes; D005942:Gluconates; D009243:NAD; D009249:NADP; C063875:2,5-diketogluconate; D013401:Sugar Alcohol Dehydrogenases; C053163:2,5-diketo-D-gluconate 5-reductase