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

  1. Gulsah Sanli1,2,
  2. Scott Banta3,†,
  3. Stephen Anderson4,
  4. Michael Blaber1,*

Article first published online: 1 JAN 2009

DOI: 10.1110/ps.03450704

Issue

Protein Science

Protein Science

Volume 13, Issue 2, pages 504–512, February 2004

Additional Information

How to Cite

Sanli, G., Banta, S., Anderson, S. and Blaber, M. (2004), Structural alteration of cofactor specificity in Corynebacterium 2,5-diketo-D-gluconic acid reductase. Protein Science, 13: 504–512. doi: 10.1110/ps.03450704

Author Information

  1. 1

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

  2. 2

    Department of Chemistry, Izmir Institute of Technology, Gulbahcekoyu-35437, Urla/Izmir, Turkey

  3. 3

    Department of Chemical and Biochemical Engineering, The State University of New Jersey, Piscataway, New Jersey 08854, USA

  4. 4

    Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA

  1. †

    Present address: Center for Engineering in Medicine, Shriners and Massachusetts General Hospitals, Harvard Medical School, Boston, MA 02114, USA.

*Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4380, USA; fax: (850) 644-7244.

Publication History

  1. Issue published online: 1 JAN 2009
  2. Article first published online: 1 JAN 2009
  3. Manuscript Revised: 10 OCT 2003
  4. Manuscript Accepted: 10 OCT 2003
  5. Manuscript Received: 19 SEP 2003

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Keywords:

  • aldo keto reductase;
  • vitamin C;
  • enzyme engineering;
  • 2,5-diketo-D-gluconic acid reductase;
  • ascorbic acid
  • and symbols: 2,5-DKG, 2,5-diketo-D-gluconic acid;
  • 2,5-DKGR, 2,5-diketo-D-gluconic acid reductase;
  • 2-KLG, 2-keto-L-gulonic acid;
  • AKR, aldo keto reductase;
  • NADH, nicotinamide adenine dinucleotide (reduced form);
  • NADPH, nicotinamide adenine dinucleotide phosphate (reduced form);
  • Tris-HCl, tris hydroxymethylaminoethane hydrochloride;
  • HEPES, N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid;
  • CD, circular dichroism;
  • GuHCl, guanidinium hydrochloride;
  • r.m.s., root mean square;
  • XR, xylose reductase

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.

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