华细辛(异)丁香酚-O-甲基转移酶(IEMT)基因的分离与功能分析  

Isolation and functional characterization of (iso)eugenol O-methyltransferase (IEMT) gene in Asarum sieboldii

作　　者：戴瑞贤 黄宝康[1,2] 刘忠 DAI Rui-xian;HUANG Bao-kang;LIU Zhong(School of Pharmacy,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China;School of Pharmacy,Naval Medical University,Shanghai 200433,China;School of Pharmaceutical Sciences,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]福建中医药大学药学院,福建福州350122 [2]海军军医大学药学院,上海200433 [3]上海交通大学药学院,上海200240

出　　处：《中国中药杂志》2024年第10期2680-2688,共9页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(32270384)。

摘　　要：甲基丁香酚(methyleugenol,ME)是中药细辛Asari Radix et Rhizoma挥发油中主要活性成分之一,具有镇痛、麻醉、抗炎等功效,其在植物体内的生物合成是初始前体物苯丙氨酸在众多酶的催化下经过一系列中间产物而完成的过程。丁香酚-O-甲基转移酶(eugenol O-methyltransferase,EOMT)是其中的关键酶之一,可使丁香酚苯环4位上的羟基甲基化,从而将丁香酚转化为甲基丁香酚。该研究首次从华细辛Asarum sieboldii中克隆1条(异)丁香酚-O-甲基转移酶[(iso)eugenol O-methyltransferase,IEMT]基因,其开放阅读框包含1 113 bp,编码1个由370个氨基酸残基组成的蛋白质;该蛋白具有甲基转移酶特征性结构域,以及二聚化结构域;构建原核表达重组质粒pET28a(+)-AsIEMT,诱导目标蛋白表达并分离纯化蛋白,以及对目标蛋白进行体外酶学分析,结果表明,AsIEMT具有双重催化活性,既能催化丁香酚生成甲基丁香酚,也能催化异丁香酚生成甲基异丁香酚,但后者效率更高;以异丁香酚为底物时,酶反应动力学常数K_(m)为(0.90±0.06)mmol·L^(-1),V_(max)为(1.32±0.04) nmol·s^(-1)·mg^(-1)。该研究拓展了对苯丙素类次生代谢关键酶基因的了解,为中药细辛品质形成机制的阐明提供了重要的研究基础。Methyleugenol is one of the main active constituents in the volatile oil of the traditional Chinese medicine Asari Radix et Rhizoma.It possesses various pharmacological effects such as analgesic,anesthetic,and anti-inflammatory properties.In biosynthesis,the initial precursor phenylalanine is finally converted into methyleugenol through a series of intermediate compounds including coniferyl acid,courmaryl acid,caffeic acid,ferulic acid/ferulic-CoA,coniferyl aldehyde,conferyl alcohol,cnfiferyl acetate,and eugenol/isoeugenol,which are produced through catalysis of a large number of enzymes.Eugenol O-methyltransferase(EOMT) is one of the key enzymes in the biosynthesis pathway,capable of methylating eugenol on the para-site hydroxyl group of the benzene ring,thereby generating methyleugenol.Here,an(iso)eugenol O-methyltransferase(IEMT) gene was cloned for the first time from Asarum siebo-ldii,holding an open reading frame that consisted of 1 113 bp and encoded a protein containing 370 amino acid residues.Bioinformatics analysis results showed that this protein was equipped with the characteristic structural domains of methyltransferases such as S-adenosylmethionine(SAM) binding sites and dimerization domains.The prokaryotic expression recombinant plasmid pET28a(+)-AsIEMT was constructed,and the candidate protein was induced and purified.In vitro enzyme assays confirmed that AsIEMT had dual functions.The enzyme could catalyze the production either of methyleugenol from eugenol or of methylisoeugenol from isoeugenol,although the latter was more prevalent.When isoeugenol was used as the substrate,the kinetics parameters K_(m) and V_(max) of catalytic reaction were(0.90±0.06) mmol·L~(-1) and(1.32±0.04)nmol·s~(-1)·mg~(-1),respectively.This study expanded our understandings of critical enzyme genes involved in phenylpropanoid metabolic pathways,and would facilitate the elucidation of quality formation mechanisms of the TCM Asari Radix et Rhizoma.

关 键 词：华细辛 甲基(异)丁香酚 O-甲基转移酶 功能鉴定 

分 类 号：S567.239[农业科学—中草药栽培]