阿维菌素起始酰基转移酶的底物特异性

Specificity of the loading acyltransferase from avermectin modular polyketide synthase

[背景]阿维菌素起始酰基转移酶(AveAT0)能够以2-甲基丁酰-辅酶A (coenzyme A,CoA)和异丁酰-CoA作为起始单元分别合成"a"系列或"b"系列的阿维菌素。[目的]探究AveAT0对两种底物的偏好性并进行改造。[方法]通过与识别不同底物的起始酰基转移酶(loading acyltransferases,AT0s)进行序列比对,找到AveAT0底物结合重要的氨基酸,利用活性位点定点突变的方法得到对底物偏好性改变的特定突变体。以2-甲基丁酰-CoA、异丁酰-CoA的类似物2-甲基丁酰-N-乙酰半胱氨(N-acetylcysteamine,SNAC)和异丁酰-SNAC为底物,用Ellman测试法检测释放SNAC的游离巯基(sulfhydryl,SH),测定AveAT0及其突变体的动力学常数,以此表征AveAT0及其突变体的底物偏好性。[结果]AveAT0对2-甲基丁酰SNAC的Km值为0.4 mmol/L,kcat值为14.1 min^-1,kcat/Km为32.1 L/(mmol·min);对异丁酰-SNAC的Km值为0.8 mmol/L,kcat值为6.4 min^-1,kcat/Km为7.5 L/(mmol·min)。选定的突变位点为V224M、Q149L、L121M。按顺序累积突变后发现三突变株AveAT0 V224M/Q149L/L121M对两个底物的偏好性区别最大,对2-甲基丁酰SNAC的Km值为0.8 mmol/L,kcat值为5.4 min^-1,kcat/Km为6.9 L/(mmol·min);对异丁酰-SNAC的kcat/Km为0.1 L/(mmol·min)。[结论]研究发现了AveAT0识别底物过程中的关键氨基酸,为改造阿维菌素聚酮合酶酰基转移酶提供了依据。

[Background] Under normal conditions in vivo,loading acyltransferase from avermectin modular polyketide synthase(AveAT0) recruits 2-methylbutyryl-coenzyme A(CoA) or isobutyryl-CoA as starter units to synthesize avermectins of series or "b" series,respectively.[Objective] We are aiming to explore the catalytic specificity of AveAT0 on 2-methylbutyry-SNAC and isobutyryl-SNAC,as substrate analogues of 2-methylbutyryl-CoA and isobutyryl-CoA and modify its favorability towards the former.[Methods] The protein sequences of several AT0 s were compared and residues playing important roles in protein-substrate identification were uncovered.The mutation experiments were then conducted on AveAT0 and concentration of free sulfhydryl(SH) released from SNAC was obtained via Ellman test to characterize the substrate specificity of mutated enzymes.[Results] The Km value of AveAT0 towards2-methylbutyry-SNAC was 0.4 mmol/L,kcat value was 14.1 min^-1 and kcat/Km value was 32.1 L/(mmol·min);the Km value of AveAT0 towards isobutyryl-SNAC was 0.8 mmol/L,kcat value was 6.4 min^-1 and the kcat/Km value was 7.5 L/(mmol·min).The mutation sites were selected as V224 M,Q149 L and L121 M.After trials in combined mutations,the specificity of a triple mutant AveAT0 V224 M/Q149 L/L121 M for both substrates increased.The Km value of AveAT0 V224 M/Q149 L/L121 M towards 2-methylbutyryl SNAC was 0.8 mmol/L,kcat value was 5.4 min^-1,and kcat/Km value was 6.9 L/(mmol·min);the kcat/Km towards isobutyryl-SNAC was 0.1 L/(mmol·min).[Conclusion] This study found key residues in AveAT0 identification,and provided insight for the rational modification of avermectin polyketide synthase acyltransferase.