The NADPH-dependent 2,5-diketo-D-gluconic acid (2,5-DKG) reductase enzyme is a required component in some novel biosynthetic vitamin C production processes. This enzyme catalyzes the conversion of 2,5-DKG to 2-keto-L-gulonic acid, which is an immediate precursor to L-ascorbic acid. Forty unique site-directed mutations were made at five residues in the cofactor-binding pocket of 2,5-DKG reductase A in an attempt to improve its ability to use NADH as a cofactor. NADH is more stable, less expensive and more prevalent in the cell than is NADPH. To the best of our knowledge, this is the first focused attempt to alter the cofactor specificity of a member of the aldo-keto reductase superfamily by engineering improved activity with NADH into the enzyme. Activity of the mutants with NADH or NADPH was assayed using activity-stained native polyacrylamide gels. Eight of the mutants at three different sites were identified as having improved activity with NADH. These mutants were purified and subjected to a kinetic characterization with NADH as a cofactor. The best mutant obtained, R238H, produced an almost 7-fold improvement in catalysis with NADH compared with the wild-type enzyme. Surprisingly, most of this catalytic improvement appeared to be due to an improvement in the apparent kcat for the reaction rather than a large improvement in the affinity of the enzyme for NADH.

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.

Total DNA of a population of uncultured organisms was extracted from soil samples, and by using PCR methods, the genes encoding two different 2,5-diketo-D-gluconic acid reductases (DKGRs) were recovered. Degenerate PCR primers based on published sequence information gave internal gene fragments homologous to known DKGRs. Nested primers specific for the internal fragments were combined with random primers to amplify flanking gene fragments from the environmental DNA, and two hypothetical full-length genes were predicted from the combined sequences. Based on these predictions, specific primers were used to amplify the two complete genes in single PCRs. These genes were cloned and expressed in Escherichia coli. The purified gene products catalyzed the reduction of 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid. Compared to previously described DKGRs isolated from Corynebacterium spp., these environmental reductases possessed some valuable properties. Both exhibited greater than 20-fold-higher k cat /K m values than those previously determined, primarily as a result of better binding of substrate. The K m values for the two new reductases were 57 and 67 M, versus 2 and 13 mM for the Corynebacterium enzymes. Both environmental DKGRs accepted NADH as well as NADPH as a cosubstrate; other DKGRs and most related aldo-keto reductases use only NADPH. In addition, one of the new reductases was more thermostable than known DKGRs.Interest in 2,5-diketo-D-gluconic acid reductases (DKGRs) is related to a new biotechnological process for the production of vitamin C (ascorbic acid) from glucose. Conversion of glucose to ascorbic acid is a complicated process that involves selective epimerization, oxidation, and lactonization reactions. The natural biosynthetic pathways are long and incorporate many energy-consuming reactions (8,21,38). The present commercial process for ascorbic acid production (the Reichstein process) couples a single biological step-the microbial oxidation of sorbitol to sorbose-with a subsequent, multistep, chemical conversion of blocked derivatives of sorbose to ascorbic acid (7,26). An alternative commercial process that has been proposed (2, 10, 34) consists of biological conversion of glucose to 2-keto-L-gulonic acid, which is then lactonized chemically to ascorbic acid (Fig. 1). The biological metabolism involved is simpler than that of natural biosynthetic routes and requires less metabolic energy (less ATP and NADPH). In this process, glucose is first converted to 2,5-diketo-D-gluconic acid by endogenous oxidases of a suitable bacterial strain, with molecular oxygen as the ultimate electron acceptor. 2,5-Diketo-D-gluconic acid is then reduced enzymatically to 2-keto-L-gulonic acid by a heterologous DKGR expressed in the production strain. The NADPH required for the reaction is generated by the metabolism of the host strain. Finally, chemical lactonization of 2-keto-L-gulonic acid generates ascorbic acid.To date, only two DKGRs have been extensively characterized, both isolated from a species of Co...

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