Molecular modeling of substrate binding in wild-type and mutant Corynebacteria 2,5-diketo-D-gluconate reductases

2,5‐Diketo‐D‐gluconic acid reductase (2,5‐DKGR; E.C. 1.1.1.‐) catalyzes the Nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent stereo‐specific reduction of 2,5‐diketo‐D‐gluconate (2,5‐DKG) to 2‐keto‐L‐gulonate (2‐KLG), a precursor in the industrial production of vitamin C (L‐ascorbate). M...

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Published in:Proteins, structure, function, and bioinformatics structure, function, and bioinformatics, 2000-04, Vol.39 (1), p.68-75
Main Authors: Khurana, Sumit, Sanli, Gulsah, Powers, David B., Anderson, Stephen, Blaber, Michael
Format: Article
Language:English
Subjects:
2,5-Diketo-D-gluconic acid
2,5-Diketo-D-gluconic acid reductase
2,5‐diketo‐D‐gluconate
2-Keto-L-gulonic acid
5-diketo-D-gluconate
aldo-keto reductase
Amino Acid Substitution
Binding Sites
Corynebacterium
Corynebacterium - enzymology
Genetic Variation
Isoenzymes - chemistry
Isoenzymes - genetics
Isoenzymes - metabolism
L-ascorbate
Models, Molecular
molecular dynamics
molecular modeling
Point Mutation
Protein Conformation
substrate recognition
Sugar Alcohol Dehydrogenases - chemistry
Sugar Alcohol Dehydrogenases - genetics
Sugar Alcohol Dehydrogenases - metabolism
Thermodynamics
vitamin C
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Summary:2,5‐Diketo‐D‐gluconic acid reductase (2,5‐DKGR; E.C. 1.1.1.‐) catalyzes the Nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent stereo‐specific reduction of 2,5‐diketo‐D‐gluconate (2,5‐DKG) to 2‐keto‐L‐gulonate (2‐KLG), a precursor in the industrial production of vitamin C (L‐ascorbate). Microorganisms that naturally ferment D‐glucose to 2,5‐DKG can be genetically modified to express the gene for 2,5‐DKGR, and thus directly produce vitamin C from D‐glucose. Two naturally occurring variants of DKGR (DKGR A and DKGR B) have been reported. DKGR B exhibits higher specific activity toward 2,5‐DKG than DKGR A; however, DKGR A exhibits a greater selectivity for this substrate and significantly higher thermal stability. Thus, a modified form of DKGR, combining desirable properties from both enzymes, would be of substantial commercial interest. In the present study we use a molecular dynamics–based approach to understand the conformational changes in DKGR A as the active site is mutated to include two active site residue changes that occur in the B form. The results indicate that the enhanced kinetic properties of the B form are due, in part, to residue substitutions in the binding pocket. These substitutions augment interactions with the substrate or alter the alignment with respect to the putative proton donor group. Proteins 2000;39:68–75. © 2000 Wiley‐Liss, Inc.
ISSN:0887-3585
1097-0134
DOI:10.1002/(SICI)1097-0134(20000401)39:1<68::AID-PROT7>3.0.CO;2-Y