赭曲霉11α羟化酶的克隆表达及关键氨基酸位点分析

Cloning and Expression of 11αHydroxylase from Aspergillus ochraceus and Analysis of Key Amino Acid Sites

11α,17α-双羟基黄体酮是甾体激素类药物的重要中间体,工业上主要利用霉菌对17α-羟基黄体酮的11α羟化反应制备。对赭曲霉的11α羟化酶及其关键氨基酸位点展开研究,为深入解析酶的催化机理提供基础数据。利用底物转化实验探究了10个羟化反应常用霉菌对17α-羟基黄体酮的转化能力,考察了赭曲霉来源的11α羟化酶CYP68J5在不同表达系统中的活性,借助结构预测、分子对接和定点突变等手段对CYP68J5的关键氨基酸位点进行解析。结果表明,赭曲霉的转化能力最强,转化时间60 h的摩尔产率达到最大值,为78.55%;其羟化酶CYP68J5在酿酒酵母中的表达活性最高;位于底物结合口袋附近的D118、F216、M488是CYP68J5的关键氨基酸位点,这些位点在维持酶的结构稳定性上发挥重要作用,是后续分子改造的潜在重要靶点。

11α,17α-dihydroxy progesterone is an important intermediate of steroid drugs,and is mainly produced by the 11αhydroxylation reaction of 17α-hydroxyprogesterone with molds in industry.In this paper,11αhydroxylase of Aspergillus ochraceus and its key amino acid sites were investigated,which can provide basic data for further analysis of the catalytic mechanism of 11αhydroxylase.Using the substrate transformation,the conversion of 17α-hydroxyprogesterone by 10 molds with hydroxylation reaction was studied,then the activity of 11αhydroxylase CYP68J5 from A.ochraceus in different expressing systems was evaluated.Finally,the key amino acid sites of CYP68J5 were analyzed by means of structure prediction,molecular docking and site-directed mutation.The results showed that A.ochraceus had the strongest conversion ability,and the maximum molar production rate of 11α,17α-dihydroxy progesterone reached 78.55%at 60 h.CYP68J5 had the highest activity in Saccharomyces cerevisiae.D118,F216 and M488 near the substrate binding pocket were identified as key amino acid sites of CYP68J5,and they played important roles in maintaining the structural stability of the enzyme and would be the potential targets for further engineering.