Structure-guided engineering of glucitol dehydrogenase cosubstrate specificity

HARTMAN MD; FIGUEROA CM; IGLESIAS AA

Mendoza

Congreso; XLVIII Reunión Anual de la SAIB; 2012

SAIB

Glucitol (Gol) is the major photosynthetic product in plants from the Rosaceae family (which includes peach, apple, and pear, among others). NAD(+)-dependent Gol dehydrogenase (GolDHase, EC 1.1.1.14) is the enzyme responsible for metabolizing this polyol in heterotrophic tissues of these plants. In contrast to its metabolic relevance, few studies deal with this enzyme and no structures have been obtained for any plant GolDHase. In this work, we built a homology model for the enzyme from peach (Prunus persica) fruits using GolDHases from Homo sapiens and Bemisia argentifolii as templates. With this model we determined the NAD(+)-binding pocket and found that Asp216 might be involved in cosubstrate specificity. Site saturation mutagenesis at position 216 and a high-throughput method for analyzing the resulting clones led us to identify several mutants capable of using NADP(+). DNA sequencing of these clones showed an Ala or a His at position 216, as key residues for determining the dinucleotide specificity. Engineering of substrate specificity on GolDHase is important due to its potential biotechnological applications, and our methodology is feasible to obtain enzymes with the desired characteristics and to reach key designs to improve the affinity for NADP(+) and other polyols.