Abstract：
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.
摘要：
甲基丁香酚是中药细辛挥发油中的主要活性成分之一。它具有多种药理作用，如镇痛，麻醉剂,和抗炎特性。在生物合成中，最初的前体苯丙氨酸最终通过一系列中间化合物转化为甲基丁香酚，包括松柏酸，Courmaryl酸,咖啡酸,阿魏酸/阿魏酸-CoA,松柏醛,conferylalcohol,乙酸丙氨酯,和丁香酚/异丁香酚，它们是通过大量酶的催化产生的。丁香酚O-甲基转移酶(EOMT)是生物合成途径中的关键酶之一,能够在苯环的对位羟基上甲基化丁香酚，从而产生甲基丁香酚。这里,首次从细辛中克隆了一个（异）丁香酚O-甲基转移酶（IEMT）基因，拥有一个由1113bp组成的开放阅读框，编码一种含有370个氨基酸残基的蛋白质。生物信息学分析结果表明，该蛋白具有甲基转移酶的特征性结构域，如S-腺苷甲硫氨酸（SAM）结合位点和二聚化结构域。构建原核表达重组质粒pET28a(+)-AsIEMT,并对候选蛋白进行诱导和纯化。体外酶测定证实AsIEMT具有双重功能。该酶可以催化丁香酚产生甲基丁香酚或异丁香酚产生甲基异丁香酚，尽管后者更为普遍。当使用异丁香酚作为底物时，催化反应动力学参数K_m和V_(max)分别为(0.90±0.06)mmol·L~(-1)和(1.32±0.04)nmol·s~(-1)·mg~(-1),分别。这项研究扩大了我们对涉及苯丙素代谢途径的关键酶基因的理解，有助于阐明中药细辛的质量形成机制。
