Study On Catalytic Hybridization Of Pueraria Glycosyltransferase

Enzymatic glycosylation catalyzed by glycosyltransferases, as a strategy of glycosylated modification, has an important effect on the creation of bioactive glycosylated small-molecule compounds. In order to obtain novel glycosyltransferases with high catalytic activity and promiscuity, we mined the glycosyltransferases from Pueraria lobata having many glycosides, and 13 new P. lobata GT genes (PlGT6-PlGT18) were successfully cloned and heterologously expressed in Escherichia coli. P1GT7 was found to possess glycosylation activity with high conversion rate on study of the glycosylated capability of PIGTs in vivo. And then, the enzymatic characteristics and catalytic promiscuity of P1GT7 has been investigated and the results are shown as follows.P1GT7 displayed maximum activity at pH 9.0 and 40℃, and was divalent-cation-independent. P1GT7 exhibited the calculated Km values of 383.3 μM for daidzein and 310.5 μM for genistein.P1GT7 exhibited the capability to glycosylate 34 structurally diverse drug-like scaffolds (such as flavonoids, lignans, alkaloids and steroid) and simple phenolics with UDP-glucose to form O-, S-, and N-glucosides. P1GT7 is the first reported plant GT having ability to catalyze alkaloids and steroid. In addition,19 out of 34 acceptors have at least two glycosylated products in the glycosylation catalyzed by P1GT7. These results demonstrated that the P1GT7 is a novel glycosyltransferase with substrate promiscuity. P1GT7 also has the reaction reversibility coupling glycosylation and deglycosylation together, which can be used to generate bioactive glycosides through one-pot reactions.18 glycosylated products from 10 aglycon acceptors were obtained via preparative-scale reactions catalyzed by P1GT7, their structures were illuminated by MS and NMR spectroscopic data analysis. The results showed that 16 glycosylated products had one glucosyl moiety,2 glycosylated products had two glucosyl moieties. The β-anomeric configuration of all glucosyl moieties was determined from the large coupling constant of the anomeric proton, and 9 glucosides of 18 glycosylated products were novel compounds.The in vitro activity of isolated glycosylated products along with its aglycons were evaluated, the results revealed that the glycosylated compound Phenylpropyl-4-β-D-glucosyloxy-2,6-dihydroxybenzoate, had a more potent neuroprotective effect than that of its corresponding aglycon and positive control drug.Based on the above results, the novel glycosyltransferase (P1GT7) with substrate promiscuity could be exploited as a useful tool enzyme for the enzymatic synthesis of structurally diverse bioactive glycosides of bioactive natural products for the discovery of drug leads or candidates. Therefore, this study of PIGTs from P. lobata is of importance both in theory and practice.