摘要
[目的]定向改造3-酮类固醇9α-羟化酶(KSH)中KshA51亚基底物结合口袋残基,催化4-雄烯二酮(4-AD)合成类固醇药物中间体7α-羟基-4雄甾烯-3,17-二酮(7α-OH AD)。[方法]同源建模预测KshA51蛋白结构,经PyMOL软件分析活性中心关键残基,合成大肠杆菌(Escherichia coli)密码子偏好性kshA51基因片段,构建野生型及突变体质粒,转化至大肠杆菌BL21细胞制备粗酶液,KshA51与KshB122共表达催化4-AD转化。高效液相色谱仪(HPLC)检测7α-OH AD保留时间处峰面积占比、筛选最佳突变体,亲和层析法纯化KshA51野生型及突变体粗酶液,SDS-PAGE法检测目的蛋白表达。[结果]KshA51最佳突变体为D311Q,其与KshB122粗酶液1∶1组合,4-AD终浓度为5 g/L,助溶剂为0.4%吐温80,转化温度为30℃,在上述催化体系中7α-OH AD转化率达55.59%,目的蛋白均成功表达。[结论]KshA51底物结合口袋关键残基突变,使KSH催化类固醇C9-羟基化活性部分转为C7-羟基化活性,为生物合成7α-OH AD提供新路径。
[Objective]Targeted modification of the KshA51 substrate binding pocket residue in 3 ketosteroid 9αhydroxylase(KSH)catalyzes the synthesis of the steroidal drug intermediate 7α-hydroxy-4 androstene-3,17-dione(7α-OH AD)from 4-androstenedione(4-AD).[Method]Homology modelling predicted the structure of KshA51 protein,and key residues in the active site were analyzed using PyMOL software;Escherichia coli codon-preferring kshA51 gene fragment was synthesized,and wild type and mutant plasmids were constructed and transformed into E.coli BL21 cells to produce crude enzyme solution,and the co-expression of KshA51 and KshB122 catalyzed the conversion of 4-AD.KshA51 and KshB122 were co-expressed to catalyze the 4-AD conversion;the best mutants were screened by high performance liquid chromatography(HPLC)with the ratio of peak area at the time of 7αOH AD;the crude enzyme solution was purified from KshA51 wild type and mutant by affinity chromatography,and the expression of target proteins was detected by SDS-PAGE.[Result]The best mutant of KshA51 was D311Q,when combined with KshB122 crude enzyme solution at 1:1 ratio,the final concentration of 4 AD was 5 g/L,the co-solvent was 0.4%Tween 80,and the conversion temperature was 30 C.The conversion rate of 7αOH AD in the above catalytic system reached 55.59%and all target proteins were successfully expressed.[Conclusion]The mutation of KshA51 substrate binding pocket residues partially converts the C9-hydroxylation activity of KSH to C7-hydroxylation activity of steroids,providing a new pathway for the biosynthesis of 7αOH AD.
作者
胡艳玲
吕德强
马倩倩
苏正定
宋士奎
HU Yanling;LYU Deqiang;MA Qianqian;SU Zhengding;SONG Shikui(School of Life and Health Sciences,Hubei University of Technology,Wuhan 430068;School of Pharmaceutical Sciences and Institute of Materia Medica,Xinjiang University,Urumqi 830046;School of Life Sciences,Xinjiang Normal University,Urumqi 830017,China)
出处
《生物技术》
2026年第1期22-29,13,共9页
Biotechnology
基金
国家自然科学基金项目(22468047)。
