Structure-Guided Engineering of Glucitol Dehydrogenase Cosubstrate Specificity

M.D. HARTMAN; C.M. FIGUEROA; A.A. IGLESIAS

Mendoza

Congreso; XLVIII Reunión Anual de la SAIB; 2012

SAIB

Glucitol (Gol) is the major photosynthetic product in plants from theRosaccac family (which includcs pca~h,a pplc, and pcar, amongnthers). NAD"dependcat Gol dehydrogcnase (ColDfIase. EC1.1.1.I4) is the enzyme mponsible for metabolizing this polyol inLeterutmphlc tissues of these plants In contras7 to its metabol~crelevance, few hidies deal with this enzyme and no structures havebeen ublained fur my p h i GulDHase. in this work, we built ahomology model for the rnzyme from peach (Prunm persiua) Fruitsusing GolDBases from Homo supiens and Bemiria argentifolii asiemplates. With this model we k t m n e d the NAD-binding pocketand found that AWalbm ight be involved in cosubstrate speciiicity.Sitc saturation rnutagmesis at position 2 1 6 and a high-throughputm&od for analyi-ing the rcsulling clones led us to identify scvcralmutants capzble of using MABP'. DNA scqucncing of these clonesshowed an Ala or a 1Tis at nosifion 216, ax kev residua fordetermining the dinucleotide specificiiy. F~nginecringo f substratesaecificitv on GolDllase is imwrtant due to its potentialb~otechnological applica:ions, and our methodology is feastble toobtain enzymes wth the desirsd charactenshcs md im reach keydes~gntso improve the affinity for NADP' and othapolyuls.