定点饱和突变提高赭曲霉11α羟化酶的催化性能

Site-directed Saturation Mutagenesis to Improve the Catalytic Performance of 11α-hydroxylase from Aspergillus ochraceus

【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。

【Objective】11α,17α-dihydroxyprogesterone is an important steroid hormone drug intermediate with high application value.The 11αhydroxylase 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 11αhydroxylase.