Cofactor specificity switch on peach glucitol dehydrogenase

HARTMAN, MATÍAS D.; MINEN, ROMINA I.; IGLESIAS, ALBERTO A.; FIGUEROA, CARLOS M.

BIOCHEMISTRY

AMER CHEMICAL SOC

Año: 2019

0006-2960

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