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Published online before print January 10, 2004, 10.1110/ps.03450704
Protein Science (2004), 13:504-512. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Structural alteration of cofactor specificity in Corynebacterium 2,5-diketo-D-gluconic acid reductase

Gulsah Sanli1,2, Scott Banta3,5, Stephen Anderson4 and Michael Blaber1

1 Kasha Laboratory, Institute of Molecular Biophysics and Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306-4380, USA
2 Department of Chemistry, Izmir Institute of Technology, Gulbahcekoyu-35437, Urla/Izmir, Turkey
3 Department of Chemical and Biochemical Engineering and
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

(RECEIVED September 19, 2003; FINAL REVISION October 10, 2003; ACCEPTED October 10, 2003)



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.

Keywords: aldo keto reductase; vitamin C; enzyme engineering; 2,5-diketo-D-gluconic acid reductase; ascorbic acid

Abbreviations: 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

Reprint requests to: Michael Blaber, Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4380, USA; e-mail: blaber{at}sb.fsu.edu; fax: (850) 644-7244.

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

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03450704.


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