催化根皮素生成三叶苷的来源于穿心莲的糖基转移酶UGT74L2及其酶学研究

The UGT74L2 of Andrographis paniculata catalyzes phloretin to produce trilobatin and its enzymatic study

根皮素-4’-O糖基转移酶(P4’-OGT)是三叶苷生物合成途径中最后一步关键酶,可在体外催化根皮素生成三叶苷,但目前仅有少数P4’-OGT被鉴定。本研究利用已报道的苹果中P4’-OGT(MdPh-4’-OGT)序列对穿心莲转录组进行筛选,获得两条同源基因UGT74L2和UGT74L3。系统发育树分析表明,UGT74L2和UGT74L3与其他物种中已鉴定功能的UGT74家族聚为一类。体外酶促反应显示,UGT74L2可特异性催化根皮素生成三叶苷,而UGT74L3无产物产生。通过Ni-NTA亲和色谱纯化获得可溶性UGT74L2重组蛋白,以根皮素为底物进行酶促动力学研究。研究结果表明,UGT74L2酶促反应最适温度为40℃,最适pH值为8.0(Tris-HCl体系)。Ca^(2+)、Mn^(2+)、Co^(2+)对UGT74L2的活性有一定的抑制作用,而Mg^(2+)可提高UGT74L2的活性,其他金属离子对UGT74L2活性无明显影响。酶促动力学参数测定结果显示,K_(m)值为29.84μmol·L^(-1),kcat值为0.02 s^(-1),k_(cat)·K_(m)^(-1)值为572.6 mol^(-1)·s_(-1)。同源建模、分子对接结合突变实验结果显示,底物结合口袋中的多个氨基酸与其催化活性密切相关。本研究从穿心莲中鉴定了一条新的P4’-OGT,可为活性天然产物三叶苷的生物合成提供糖基化元件,也可为其他植物糖基转移酶的功能挖掘提供参考。

The last essential enzyme in the biosynthetic pathway of trilobatin,phloretin-4’-O glycosyltransferase(P4’-OGT),catalyzes the conversion of trilobatin to phloretin in vitro.However,only a few P4’-OGTs have been found in plants.This study used Malus domestica phloretin-4’-O glycosyltransferase(MdPh-4’-OGT)as a query to identify and clone two UDP-glucuronosyltransferase(UGT)genes,designated UGT74L2 and UGT74L3,from the transcriptome of Andrographis paniculata.According to a phylogenetic tree analysis,UGT74L2 and UGT74L3 belonged to the UGT74 family,which has been linked to several activities in other species.The in vitro enzymatic reaction demonstrated that UGT74L2 could particularly catalyze the formation of trilobatin from phloretin,but UGT74L3 had no effects.By using Ni-NTA affinity chromatography to extract the soluble UGT74L2 recombinant protein,the enzymatic kinetics of the activity was investigated using phloretin as the substrate.The results showed that the optimal temperature and pH for UGT74L2 enzymatic reaction were 40℃and 8.0(Tris-HCl system),respectively.Three metal ions(Ca^(2+),Mn^(2+)and Co^(2+))showed inhibitory effect on the activity of UGT74L2,while Mg^(2+)could improve the activity of UGT74L2.Other tested metal ions have no significant effect on UGT74L2.The results of enzymatic kinetic parameters that the K_(m)value was 29.84μmol·L_(-1),the k_(cat)was 0.02 s^(-1),and the k_(cat)·K_(m)^(-1)was 572.6 mol^(-1)·s^(-1).By homology modeling,molecular docking and mutation experiments,we found that multiple amino acids residues around the substrate binding pocket play quite an important role during catalytic process,In summary,we identified a novel P4’-OGT gene from medicinal plant Andrographis paniculata and provided a new efficient catalyst to synthesize trilobatin.Meanwhile,this study provides a reference for mining new efficient glycosylation modules from plants.