Regioselective glucosidation of trans-resveratrol in Escherichia coli expressing glucosyltransferase from Phytolacca americana

Abstract

A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-β-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4'-O-β-glucoside: 3-O-β-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue.

Scheme 1

Glucosidation of trans-resveratrol

Fig. 1

HPLC profiles of the glucosylated products obtained from incubation with a Phytolacca americana cells in the absence of auxin, b P. americana cells in the presence of 2,4-dichlorophenoxyacetic acid, and c the purified PaGT3 enzyme. * and ** indicate trans-resveratrol 4′-O-β-glucoside and trans-resveratrol 3-O-β-glucoside, respectively. d The starting material trans-resveratrol was eluted at 31 min

Fig. 2

HPLC profiles of glucosylated products obtained from the incubation of E. coli expressing a wild type PaGT3 gene and b H20A PaGT3 gene. HPLC profile b was similar to that obtained in the incubation with E. coli expressing H20D PaGT3 gene. *, **, and *** indicate trans-resveratrol 4′-O-β-glucoside, trans-resveratrol 3-O-β-glucoside, and trans-resveratrol, respectively

Fig. 3

a The active site of PaGT3 model. The predicted PaGT3 structure was superimposed upon the crystal structure of VvGT bearing UDP-2-deoxy-2-fluoro glucose (nontransferable donor) and kaempferol (acceptor). UDP-2-deoxy-2-fluoro glucose (U), kaempferol (K), and some selected active site residues of PaGT3 are shown. b Schematic diagram of active site of PaGT3 model. The dashed lines indicate hydrogen bonds. A hydrogen bond between His-20 and the 4′-hydroxy group of trans-resveratrol may promote nucleophilic attack on C1 of UDP-glucose to produce 4′-β-O-glucoside

Fig. 4

SDS-PAGE of the purified 1 H20A and 2 H20D PaGT3 enzymes