Cofactor Specificity Switch on Peach Glucitol Dehydrogenase

• DOI: 10.1021/acs.biochem.8b01240
• Publication Date: 2019-02-06T21:36:21Z
• Authors: Matías D. Hartman; Romina I. Minen; Alberto A. Iglesias; Carlos M. Figueroa
• Keywords: Saturated mutagenesis; Site-directed mutagenesis

Most oxidoreductases that use NAD+ or NADP+ to transfer electrons in redox reactions display a strong preference for the cofactor. The catalytic efficiency of peach glucitol dehydrogenase (GolDHase) is 1800-fold higher than NADP+. Herein, we combined structural and kinetic data reverse cofactor specificity this enzyme. Using site-saturation mutagenesis, obtained D216A mutant, which uses both NADP+, although with different efficiencies (1000 ± 200 170 30 M–1 s–1, respectively). This mutant was used as template introduce further mutations by site-directed using information from fruit fly NADP-dependent GolDHase. D216A/V217R/D218S triple displayed 2-fold NAD+. Overall, our results indicate has potential be metabolic cellular engineering recycling industrial processes.