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

Gulsah Sanli, Scott Banta, Stephen Anderson, Michael Blaber

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

Carynebacterium 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.

Original languageEnglish (US)
Pages (from-to)504-512
Number of pages9
JournalProtein Science
Volume13
Issue number2
DOIs
StatePublished - Feb 2004

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Keywords

  • 2,5-diketo-D-gluconic acid reductase
  • Aldo keto reductase
  • Ascorbic acid
  • Enzyme engineering
  • Vitamin C

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