文献类型：期刊文献

中文题名：基于长链底物亲和的脂肪酶MAS1理性设计改造

英文题名：Rational Design of Lipase MAS1 for the Long-Chain Substrate Affinity

作者：唐秀云[1];王嘉伟[2];张建国[2];缪雨露[3];赵玥[3];高蓓[1];张鲁嘉[1,3,4]

机构：[1]华东理工大学生物工程学院,生物反应器工程国家重点实验室,上海200237;[2]上海理工大学健康科学与工程学院,上海200093;[3]华东师范大学化学与分子工程学院,上海分子治疗与新药创制工程技术研究中心,上海200241;[4]纽约大学(上海)理论与计算化学联合中心,上海200122

年份：2023

卷号：49

期号：4

起止页码：529

中文期刊名：华东理工大学学报（自然科学版）

外文期刊名：Journal of East China University of Science and Technology

收录：Scopus;北大核心:【北大核心2020】；CSCD:【CSCD_E2023_2024】；

基金：国家重点研发计划(2020YFA0908400);国家自然科学基金(31772007,U1805235);上海市自然科学基金(21ZR1416500);纽约大学全球种子基金;纽约大学-华东师范大学计算化学中心(ECNU创新001公众平台)。

语种：中文

中文关键词：脂肪酶MAS1;长链底物;动力学;MM/GBSA;热点残基

外文关键词：lipase MAS1;long-chain substrate;kinetics;MM/GBSA;hotspot residues

摘要：探讨了脂肪酶MAS1对长链底物4-硝基苯酚豆蔻肉酸酯(pNP-C14)催化的最适温度、最适pH、pH稳定性,并通过分子动力学模拟计算得出结合自由能,然后根据结合自由能的差值初步筛选出了G145W及T141L两个单点突变体。实验表明,与野生型MAS1相比,结合自由能降低显著的突变体G145W的米氏常数(Km)减小了11%,催化常数(kcat)与Km的比值(kcat/Km)为野生型MAS1的1.29倍,突变体G145W对长链底物的亲和能力和催化效率均有提高;而结合自由能降低较小的突变体T141L的Km值增大了22%,kcat/Km为野生型MAS1的0.88倍,说明酶与底物分子结合自由能的降低并非准确的突变筛选标准。通过分子力学/广义波恩表面积(MM/GBSA)残基拆解分析得出:残基T38、F39、L149、F153、V202、V233对脂肪酶活性口袋与长链底物结合的稳定性贡献较其他残基大;残基T38、G40、N41、N45和T237对提高长链底物的亲和能力具有重要贡献,预测为热点残基,其结合自由能的降低可以作为突变筛选的参考标准。
The optimum temperature,pH,and pH stability of lipase MAS1 for the catalysis of the long-chain substrate 4-nitrophenol myristate(pNP-C14)were determined.Two single point mutations,G145W and T141L,were initially screened out according to the decrease of binding free energy by molecular dynamics simulation(Molecular Dynamics,MD).Experiments showed that Km of the mutant G145W with a significant decrease in binding free energy was reduced by 11%compared with the wild-type MAS1,and kcat/Km was 1.29 times that of the wild-type MAS1.Compared with the wild-type MAS1,Km of the mutant T141L with a smaller decrease in binding free energy was increased by 22%,and kcat/Km was 0.88 times that of the wild-type MAS1.Compared with the wild type,the affinity and catalytic efficiency of G145W for long-chain substrates were improved,while those of T141L were lower than the wild type.It showed that the absolute zero value of the binding energy difference was not an accurate mutation screening criterion.In-depth analysis was performed by Molecular Mechanics/Generalized Born Surface Area(MM/GBSA)residue disassembly.It was concluded that residues T38,F39,L149,F153,V202 and V233 contributed more to the binding stability of the lipase active pocket and long-chain substrates than other residues;Residues T38,G40,N41,N45,and T237 showed important contributions to the improvement of lipase affinity for long-chain substrates and were predicted to be hotspot residues whose reduced binding free energy can serve as a reference standard for mutation screening.