新型酰基转移酶AT-EF080951的重组表达及底物结合分析  

Recombinant expression of a new acyltransferase AT-EF080951 and analysis on its substrate specificity

作　　者：焦豫良[1] 王梁华[1] 董晓毅[1] 宗英[1] 高云[1] 任娜[1] 郭爱芸[1] 张兴群[1] 焦炳华[1] 

机构地区：[1]第二军医大学基础医学部,上海200433

出　　处：《中国抗生素杂志》2008年第3期140-144,共5页Chinese Journal of Antibiotics

基　　金：国家自然科学基金项目30670043;863计划海洋技术领域专题项目2006AA09Z434资助

摘　　要：目的研究新型聚酮合酶(PKS)基因簇EF568935(GenBank登录号)中酰基转移酶(AT)EF080951(GenBank登录号)底物特异性,为PKS的功能研究及组合生物合成研究提供新组件。方法从酰基转移酶AT-EF080951结构域两端的连接肽区设计引物,通过PCR克隆其结构域基因at-EF080951;连接入原核表达载体pMAL-c2X,与麦芽糖结合蛋白(MBP)融合表达;直链淀粉树脂柱亲和层析纯化MBP-AT-EF080951;融合蛋白用Xa因子切除MBP,得到AT-EF080951单体;以MBP、小牛血清白蛋白(BSA)为对照蛋白,用紫外分光光度法测定MBP-AT-EF080951与AT-EF080951对底物的结合能力,计算结合常数(Ka,μmol/L),分析其结合底物的特异性。结果(1)MBP-AT-EF080951与底物的结合常数为:甲基丙二酰辅酶A,0.0049±0.001;丙二酰辅酶A,0.0049±0.003;乙酰辅酶A,0.107±0.002;辅酶A,0.005±0.003。(2)AT-EF080951与底物的结合常数为:甲基丙二酰辅酶A,0.005±0.002;丙二酰辅酶A,0.0041±0.002;乙酰辅酶A,0.120±0.001;辅酶A,0.0042±0.003。结论MBP-AT-EF080951和AT-EF080951都特异性结合乙酰辅酶A,AT-EF080951可能是乙酰转移酶。Objectives To study the substrate specificity of a new acyhraferase （GenBank accession no. EF080951） structural domain in PKS gene cluster （GenBank accession no. EF568935） for contributing a new element for combinatorial biosynthesis. Methods at-EF080951 gene of acyhraferase AT-EF080951 was cloned through PCR by designing primers targeting at linker region sequences adjoining to both sides of the domain and inserted into prokaryotic expression vector pMAL-c2X and expressed as an MBP （maltose-binding protein） fusion protein; fusion protein MBP-AT-EF080951was purified by amylose resin affinity chromatography; MBP-AT-EF080951 was cleaved by Factor Xa to get single AT-EF080951; UV spectrophotometry was used to study the binding ability of MBP-AT-EF080951 and AT-EF080951 to substrates; binding constants（Ka, μmol/L）were calculated and used to analyze substrate specificity of it. Results （1）The binding constant of MBP-AT-EF080951 for methylmalonyl coenzyme A, 0. 0049±0.001; malonyl coenzyme A, 0.0049 ±0. 003; acetyl coenzyme A, 0. 107 ±0. 002; coenzyme A, 0. 005 ± 0. 003. （2） The binding constant of AT-EF080951 for methylmalonyl coenzyme A, 0.005 ± 0.002; malonyl coenzyme A, 0. 0041 ±0. 002; acetyl coenzyme A, 0. 120 ±0. 001; coenzyme A, 0. 0042 ±0. 003. Conclusions Among the 4 substrates, MBP-AT-EF080951 and AT-EF080951 binds to acetyl coenzyme a specifically, while AT-EF080951 is presumably an acetyltransferase from a new PKS gene cluster.

关 键 词：聚酮合酶 酰基转移酶 乙酰基转移酶 结合常数 组合生物合成 

分 类 号：Q78[生物学—分子生物学]