Shifting cofactor specificity of peach glucitol dehydrogenase by structure-guided mutagenesis

HARTMAN MD; FIGUEROA CM; MINEN RI; IGLESIAS AA

Córdoba

Congreso; LII Reunión Anual de la SAIB; 2016

SAIB

Dehydrogenases use nicotinamide adenine dinucleotides as cofactors. Thestructural domain for NAD(P)+ binding is composed of three b-strands separated by two a-helices. Based on homology modelling studies wehypothesised that Asp216 (located at the end of the second b-strand) determines the marked preference of peach (Prunuspersica) glucitol dehydrogenase forNAD+. Site-saturation mutagenesis of Asp216 to Ala increased (35-fold)the catalytic efficiency for NADP+ and decreased (9‑fold) it for NAD+. Interestingly, the triplemutant D216A/V217R/D218S (designed by comparison with a NADP+-dependentdehydrogenase) showed a catalytic efficiency for NADP+ 530-foldhigher than the wild type. Moreover, the triple mutant exhibited a catalyticefficiency for NADP+ 2-fold higher than for NAD+. Our resultsshow that both strategies produced enzymes that preferably catalyze glucitol oxidationin a NADP+‑dependent manner. These techniques could be useful toproduce novel specificities in enzymes with broad applications in biotechnologicalprocesses.