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 Phe(148) to Val, or Tyr(202) 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.