首页|定点饱和突变提高赭曲霉11α羟化酶的催化性能

定点饱和突变提高赭曲霉11α羟化酶的催化性能

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[目的]11α,17α-双羟基黄体酮是重要的甾体激素类药物中间体,应用价值大.赭曲霉(Aspergillus ochraceus)CICC 41473 的 11α羟化酶CYP68J5 是转化 17α-羟基黄体酮生成 11α,17α-双羟基黄体酮的关键酶,但其催化性能有待于提升.[方法]在课题组前期确定的关键氨基酸位点D118、F216 和M488 基础上,通过定点饱和突变和底物转化实验进行优良突变体的筛选;使用AutoDock进行酶与底物的分子对接,并通过Discovery Studio和Gromacs分别研究分子间相互作用力和分子动力学模拟.[结果]突变体D118V、F216W、M488L和M488W具有催化性能,其中,优良突变体D118V的活性最高,其在底物浓度 0.5 g/L时,生产强度为 431.66 mg/(L·d),较野生型提高了 2.12 倍,这可能是因为当 118 位的天冬氨酸突变为缬氨酸后,该位点与底物之间产生了新的疏水相互作用,增强了酶的底物结合口袋的疏水性.分子动力学模拟结果表明酶的整体构象更稳定,酶与底物的结合更紧密.[结论]通过定点饱和突变获得了催化性能显著提升的优良突变体D118V,研究结果为甾体 11α羟化酶的改造提供了应用案例和理论指导.
Site-directed Saturation Mutagenesis to Improve the Catalytic Performance of 11α-hydroxylase from Aspergillus ochraceus
[Objective]11α,17α-dihydroxyprogesterone is an important steroid hormone drug intermediate with high application value.The 11α hydroxylase CYP68J5 from Aspergillus ochraceus CICC 41473 is a key enzyme for the bioconversion from 17α-hydroxyprogesterone to 11α,17α-dihydroxyprogesterone,but its catalytic performance needs to be further improved.[Methods]Based on the key amino acid sites D118,F216 and M488 identified in our previous works,the excellent mutants were screened by site-directed saturation mutagenesis and substrate transformation experiments.Molecular docking between enzymes and substrates was performed using AutoDock,and the intermolecular interaction forces and molecular dynamic simulations were studied by Discovery Studio and Gromacs,respectively.[Results]Mutants D118V,F216W,M488L,and M488W possessed catalytic properties,among which,D118V showed the highest activity.At a substrate concentration of 0.5 g/L,the production intensity of D118V was 431.66 mg/(L·d),which was 2.12-fold higher than that of the wild type CYP68J5.This might be attributed to the fact that a new hydrophobic interaction was generated between the site and substrate when the aspartic acid at position 118 was changed to valine,consequently enhancing the hydrophobicity of the substrate-binding pocket of the enzyme.Molecular dynamics simulations showed more stable overall conformation of the mutant and tighter binding effect between enzyme and substrate.[Conclusion]The excellent mutant D118V with significantly improved catalytic performance was obtained by site-directed saturation mutagenesis.The results provide an application case and theoretical guidance for the molecular modification of steroid 11α hydroxylase.

11α,17α-dihydroxyprogesterone11α hydroxylasesite-directed saturation mutagenesismolecular dockingmolecular dynamics simulations

史京辉、陈文慧、陆坤、郑婷婷、任志远、鲍国庆、王敏、骆健美

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工业发酵微生物教育部重点实验室(天津科技大学),天津市工业微生物重点实验室 天津市微生物代谢与发酵过程控制技术工程中心,天津科技大学生物工程学院,天津 300457

11α,17α-双羟基黄体酮 11α羟化酶 定点饱和突变 分子对接 分子动力学模拟

国家自然科学基金面上项目国家自然科学基金面上项目国家合成生物学重点研发计划项目国家级大学生创新创业训练计划资助项目

21978220322701352019YFA0905300202210057045

2024

10.13560/j.cnki.biotech.bull.1985.2023-0751

生物技术通报
中国农业科学院农业信息研究所

生物技术通报

CSTPCD北大核心
影响因子：0.505
ISSN：1002-5464
年,卷(期)：2024.40(1)
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