Molecular Modeling of Substrate Binding in Wild-Type and Mutant Corynebacteria 2,5-Diketo-D-gluconate Reductases

ABSTRACT 2,5-Diketo-D-gluconic acid reduc-tase (2,5-DKGR; E.C. 1.1.1.-) catalyzes the Nicotin-amide 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 fer-ment D-glucose to 2,5-DKG can be genetically modi-fied 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 proper-ties of the B form are due, in part, to residue substitutions in the binding pocket. These substitu-tions 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. Key words: molecular dynamics; molecular model-ing; aldo-keto reductase; vitamin C; substrate recognition; 2,5-diketo-D-glu-conate; L-ascorbat