Mutational analysis of the Medicago glycosyltransferase UGT71G1 reveals residues that control regioselectivity for (iso)flavonoid glycosylation.

The plant glycosyltransferase UGT71G1 from the model legume barrel medic (Medicago truncatula) glycosylates flavonoids, isoflavonoids, and triterpenes. It can transfer glucose to each of the five hydroxyl groups of the flavonol quercetin, with the 3'-O-glucoside as the major product, and to the A-ring 7-hydroxyl of the isoflavone genistein. The sugar donor and acceptor binding pockets are located in the N and C termini, respectively, of the recently determined crystal structure of UGT71G1. The residues forming the binding pockets of UGT71G1 were systematically altered by site-directed mutagenesis. Mutation of Phe148 to Val, or Tyr202 to Ala, drastically changed the regioselectivity for quercetin glycosylation from predominantly the 3'-O-position of the B-ring to the 3-O-position of the C ring. The Y202A mutant exhibited comparable catalytic efficiency with quercetin to the wild-type enzyme, whereas efficiency was reduced 3-4-fold in the F148V mutant. The Y202A mutant gained the ability to glycosylate the 5-hydroxyl of genistein. Additional mutations affected the relative specificities for the sugar donors UDP-galactose and UDP-glucuronic acid, although UDP-glucose was always preferred. The results are discussed in relation to the design of novel biocatalysts for production of therapeutic flavonoids.