EVOLUTION-GUIDED OPTIMIZATION FOR SELECTING SITEDIRECTED MUTAGENESIS SITES OF AGROBACTERIUM TUMEFACIENS URONATE DEHYDROGENASEMURUGAN R., PRATHIVIRAJ R., DIPTI MOTHAY AND CHELLAPANDI P.Abstract Uronate dehydrogenase (EC:1.1.1.203) belongs to the NAD-dependent epimerase/dehydratase (NDE/D) subfamily, which converts D-galacturonic acid and D-glucuronic acid into D-galactaric acid and D-glucaric acid, respectively. Uronate dehydrogenase-catalyzed reaction is reversible and no substratespecific activity in nature. Therefore, evolution-guided optimization approach was employed for screening, selection, and evaluation of its mutants to increase the substrate specificity with NAD+ and D-glucuronic acid. The phylogenetic analysis described that uronate dehydrogenase from A. tumefaciens evolved from the UDP-glucose-4-epimerase subfamily members and not related to closely related soil bacteria. Molecular conservation of its sequence-structure-function integrity was retained in this organism by imposing purifying selection and amino acid substitution patterns. A single amino acid substitution in its proton relay system or substrate-binding site found to bring several changes in the local structural environments. It hasenforced to optimize the substrate-binding site that recognizes the D-galacturonic acid or D-glucuronic acid. Hence, site-directed mutagenesis targets detectedinthis study would be useful for engineering uronate dehydrogenase subjected to be used in the biotransformation process of D-glucaric acid production. |