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-specif ic reduction of 2,5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonate (2-KL G), a precursor in the industrial production of vitamin C (L-ascorbate). Mi croorganisms that naturally ferment D-glucose to 2,5-DKG; can be geneticall y modified to express the gene for 2,5-DKGR, and thus directly produce vita min 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 th is substrate and significantly higher thermal stability. Thus, a modified f orm of DKGR, combining desirable properties from both enzymes, would be of substantial commercial interest. In the present study we use a molecular dy namics-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 proper ties of the B form are due, in part, to residue substitutions in the bindin g pocket. These substitutions augment interactions with the substrate or al ter the alignment with respect to the putative proton donor group. (C) 2000 Wiley-Liss, Inc.