Optimizing an Artificial Metabolic Pathway:  Engineering the Cofactor Specificity of Corynebacterium 2,5-Diketo-d-gluconic Acid Reductase for Use in Vitamin C Biosynthesis

Abstract

The strict cofactor specificity of many enzymes can potentially become a liability when these enzymes are to be employed in an artificial metabolic pathway. The preference for NADPH over NADH exhibited by the Corynebacterium 2,5-diketo-D-gluconic acid (2,5-DKG) reductase may not be ideal for use in industrial scale vitamin C biosynthesis. We have previously reported making a number of site-directed mutations at five sites located in the cofactor-binding pocket that interact with the 2'-phosphate group of NADPH. These mutations conferred greater activity with NADH upon the Corynebacterium 2,5-DKG reductase [Banta, S., Swanson, B. A., Wu, S., Jarnagin, A., and Anderson, S. (2002) Protein Eng. 15, 131-140; (1)]. The best of these mutations have now been combined to see if further improvements can be obtained. In addition, several chimeric mutants have been produced that contain the same residues as are found in other members of the aldo-keto reductase superfamily that are naturally able to use NADH as a cofactor. The most active mutants obtained in this work were also combined with a previously reported substrate-binding pocket double mutant, F22Y/A272G. Mutant activity was assayed using activity-stained native polyacrylamide gels. Superior mutants were purified and subjected to a simplified kinetic analysis. The simplified kinetic analysis was extended for the most active mutants in order to obtain the kinetic parameters in the full-ordered bi bi rate equation in the absence of products, with both NADH and NADPH as cofactors. The best mutant 2,5-DKG reductase produced in this work was the F22Y/K232G/R238H/A272G quadruple mutant, which exhibits activity with NADH that is more than 2 orders of magnitude higher than that of the wild-type enzyme, and it retains a high level of activity with NADPH. This new 2,5-DKG reductase may be a valuable new catalyst for use in vitamin C biosynthesis.

Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases

“…An effort to engineer an NADH-specific 2,5-diketo-D-gluconic acid reductase (AKR5C) from Corynebacterium for commercial purposes has yielded a quadruple mutant enzyme that improves NADH utilization by two orders of magnitude [11]. The mutations, Phe 22 → Tyr, Lys 232 → Gly, Arg 238 → His and Ala 272 → Gly are widely distributed about the dinucleotide-binding site and none would appear to facilitate the introduction of a carboxylate residue to interact with the adenosine 2 -and 3 -hydroxy groups.…”

“…7-fold for NADH [11]. The effects of mutations on residues making salt links with the 2 -phosphate have also been examined in AR [12] and human 3α-hydroxysteroid dehydrogenase [13,14].…”

Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior

“…The on-rate of the substrate with NADH decreased almost fivefold in the double mutant versus the wild-type, but increased eightfold with NADPH. The ratio of k ss 1 k ss 3 =k ss 2 is a convenient single parameter for examining the catalytic performance of the mutants (Banta et al, 2002c). When judged by this composite rate constant, the double mutant enzyme is shown to be substantially improved with NADP þ in the oxidative direction and with both NADH and NADPH in the reductive reaction compared to the wild-type (Fig.…”

Microbial aldo-keto reductases

“…2,5-diketo-D-gluconic acid reductase (AKR5C) for use in vitamin C biosynthesis, which on an industrial scale would be more economically viable if the enzyme could utilize NADH instead of NADPH [51].…”