1. Cofactor Specificity Switch on Peach Glucitol Dehydrogenase.
- Author
-
Hartman MD, Minen RI, Iglesias AA, and Figueroa CM
- Subjects
- Kinetics, L-Iditol 2-Dehydrogenase chemistry, L-Iditol 2-Dehydrogenase genetics, Mutagenesis, Site-Directed, Plant Proteins chemistry, Plant Proteins genetics, Coenzymes metabolism, L-Iditol 2-Dehydrogenase metabolism, NADP metabolism, Plant Proteins metabolism, Prunus persica enzymology
- 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.- Published
- 2019
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