Alteration of the specificity of the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase A

Abstract

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.

Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis

“…However, mutations equivalent to K270G and R276H in PsXR lead to efficient NADH preference (Banta et al, 2002a, b).…”

Production of biocommodities and bioelectricity by cell‐free synthetic enzymatic pathway biotransformations: Challenges and opportunities

“…Enzymes can be further engineered to enhance their catalytic efficiency for industrial applications, such as thermostability (Given et al, 1998;Liu et al, 2009), cofactor preference (Banta et al, 2002a;Woodyer et al, 2003), and so on, by rational design, directed evolution, or a combination (Luetz et al, 2008;Zhang et al, 2006).…”

Beyond Protein Engineering: its Applications in Synthetic Biology

“…The same team later changed the product specificity of the [+]-δ-cadinene synthase from Gossypium arboretum to create an entirely novel sesquiterpene synthase that produces germacrene D-4-ol from FPP, in order to meet the requirement of a synthetic pathway [7]. [9]. In addition, NADH is more stable than NADPH, and is also roughly an order of magnitude less expensive.…”

“…To optimize the vitamin C biosynthetic pathway, Banta et al [9] switched the cofactor preference of Corynebacterium 2,5-Diketo-D-gluconic acid [2, reductase from NADPH to NADH. The authors first subjected the binding site of the 2'-phosphate group of NADPH to site-directed mutagenesis [3].…”