296位氨基酸位阻效应影响黄花蒿没药醇合酶环化反应的起始

摘要

[Objective]This study is to explore the mechanism of inhibition on the cyclization reaction of Artemisia annua bisabolol synthase T296 mutants.[Methods]The mutation from threonine to isoleucine at position 296 of Artemisia annua bisabolol synthase was performed using QuikChange(R) Multi Site-directed mutagenesis method.Mutant protein was expressed in E.coli and purified by Ni2+ affinity chromatography and the catalytic specificity was characterized by GC and GCMS.[Results]Artemisia annua bisabolol synthase T296I mutant catalyzes (2E,6E)-farnesyl diphosphate mainly into farnesene,but incubating T296I mutant with R-nerolidyl pyrophosphate resulted in the formation of α-bisabolol,the native product of wild enzyme.[Conclusion]The volume and stereochemistry of the 296 amino acid residue side chain of Artemisia annua bisabolol synthase are key factors in the initiation of the cyclization reaction.Its steric effect inhibits the conversion of farnesyl pyrophosphate into nerolidyl pyrophosphate.%[目的]探究黄花蒿没药醇合酶第296位氨基酸突变抑制环化反应的机制.[方法]利用Quik Change(R)Multi Site-Directed Mutagenesis的方法将黄花蒿没药醇合酶的296位氨基酸残基由苏氨酸突变成异亮氨酸.原核表达,蛋白纯化后测定其催化特异性.[结果]黄花蒿没药醇合酶T296I体外催化(2E,6E)-法尼基焦磷酸主产物为非环化的法尼烯;但是将T296I蛋白与中间体(3R,6E)-橙花叔醇焦磷酸一起孵育时可生成环化的主产物α-bisabolol,野生型酶的天然产物.[结论]黄花蒿没药醇合酶T296I点突变进一步证明氨基酸残基的空间体积和立体化学是自然环化反应起始的控制关键,其位阻效应能够抑制法尼基焦磷酸向橙花叔醇焦磷酸转化.