PMID: 11917149Mar 28, 2002Paper

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

Protein Engineering
Scott BantaStephen Anderson

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...Continue Reading

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Citations

Jan 10, 2012·Chemical Communications : Chem Comm·Elliot CampbellScott Banta
May 22, 2004·European Journal of Biochemistry·Nanne M KamerbeekDick B Janssen
Aug 21, 2009·Protein Science : a Publication of the Protein Society·George A KhouryCostas D Maranas
Mar 11, 2020·International Journal of Molecular Sciences·Xiuzhen GaoMingfei Liu
Oct 14, 2016·Chembiochem : a European Journal of Chemical Biology·Andy BeierUwe T Bornscheuer
May 19, 2019·Applied Microbiology and Biotechnology·Yirong XuHairong Cheng
Jan 11, 2018·Synthetic and Systems Biotechnology·Chun YouYi-Heng Percival Zhang

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