Cofactor Specificity Switch on Peach Glucitol Dehydrogenase

Abstract

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