Probing the role of highly conserved residues forming the acceptor binding pocket of the promiscuous glycosyltransferase MGT in defining the specificity towards a panel of flavonoids.

MGT, a macrolide UDP-glycosyltransferase from Streptomyces lividans, has been employed as a synthetic "tool kit" to synthesize a series of modified antibiotics owing to its broad substrate plasticity. Other than MGT, a number of UDP-glycosyltransferases with substrate promiscuity were also used to alter glycosylation patterns of secondary metabolites in an emerging method called "chemoenzymatic glycorandomization". However, the structural basis of tolerating variant acceptors for these glycosyltransferases is still not clear. In this study, the relationship between the amino acid residues forming the acceptor binding pocket and the specificity of an MGT was investigated in evolutionary and structural aspects. Interestingly, alterations of the volume and hydrophobic environment of the binding pocket by replacing Ile127 or Val151 with a bulky Phe conferred on the MGT novel activities for glycosylating flavonoids that are not accepted by the wild-type MGT.