Analysis of the Effect of N555 Mutations on the Product Specificity of Dextransucrase Using Caffeic Acid Phenethyl Ester as an Acceptor Substrate.

Bioglycosylation is an efficient strategy to improve biological activities and physicochemical properties of natural compounds to develop structural modifications of drugs. In this study, an N555 residue was identified as a candidate for site-directed mutagenesis through sequence alignment with GTF180ΔN. Caffeic acid phenethyl ester (CAPE) was used as an acceptor substrate. Two generated mutants, N555Q and N555E, demonstrated significant specificity of distribution of products. Under identical conditions, the conversion rates of diglycoside products (CAPE-2G) generated by the N555E (80.8%) and N555Q (84.5%) mutants were 3.30- and 3.46-fold higher than those generated by the original enzyme (24.4%). The structural simulation results demonstrated that a new hydrogen bond was formed between the N555 residue and CAPE, and the N555 residue was closely related to substrate elongation. These results provide a reference for subsequent studies. Suitable mutants for transfer of diglycosides have important application potential in the food and pharmaceutical industries.