A kinetic analysis of regiospecific glucosylation by two glycosyltransferases of Arabidopsis thaliana - Domain swapping to introduce new activities

摘要

Plant Family 1 glycosyltransferases (GTs) recognize a wide range of natural and non-natural scaffolds and have considerable potential as biocatalysts for the synthesis of small molecule glycosides. Regiospecificity of glycosylation is an important property, given that many acceptors have multiple potential glycosylation sites. This study has used a domain-swapping approach to explore the determinants of regiospecific glycosylation of two GTs of Arabidopsis thaliana, UGT74F1 and UGT74F2. The flavonoid quercetin was used as a model acceptor, providing five potential sites for O-glycosylation by the two GTs. As is commonly found for many plant GTs, both of these enzymes produce distinct multiple glycosides of quercetin. A high performance liquid chromatography method has been established to perform detailed steady-state kinetic analyses of these concurrent reactions. These data show the influence of each parameter in determining a GT product formation profile toward quercetin. Interestingly, construction and kinetic analyses of a series of UGT74F1/F2 chimeras have revealed that mutating a single amino acid distal to the active site, Asn-142, can lead to the development of a new GT with a more constrained regiospecificity. This ability to form the 4'-O-glucoside of quercetin is transferable to other flavonoid scaffolds and provides a basis for preparative scale production of flavonoid 4'-O-glucosides through the use of whole-cell biocatalysis.