电子转移途径中芳香族氨基酸的引入提高了细胞色素P450s的催化性能  

Introduction of aromatic amino acids in electron transfer pathways yielded improved catalytic performance of cytochrome P450s

作　　者：孟帅奇 李忠玉 季宇 Anna Joelle Ruff 刘珞[2] Mehdi D.Davari Ulrich Schwaneberg Shuaiqi Meng;Zhongyu Li;Yu Ji;Anna Joelle Ruff;Luo Liu;Mehdi D.Davari;Ulrich Schwaneberg(Institute of Biotechnology,RWTH Aachen University,Worringerweg 3,52074 Aachen,Germany;Beijing Bioprocess Key Laboratory,Beijing University of Chemical Technology,Beijing 100029,China;Department of Bioorganic Chemistry,Leibniz Institute of Plant Biochemistry,Weinberg 3,Halle06120,Germany;DWI-Leibniz Institute for Interactive Materials,Forckenbeckstraße 50,Aachen52074,Germany)

机构地区：[1]亚琛工业大学生物技术研究所,德国 [2]北京化工大学北京生物过程重点实验室,北京100029 [3]莱布尼茨植物生物化学研究所生物有机化学系,德国 [4]莱布尼茨互动材料研究所,德国

出　　处：《Chinese Journal of Catalysis》2023年第6期81-90,共10页催化学报（英文）

基　　金：中国留学基金委员会(201906880011).

摘　　要：细胞色素P450s是用于生物合成的多功能催化剂.在P450催化循环中,需要两个电子来还原血红素铁,并通过电子转移途径(eTPs)激活随后的还原,该步骤是反应的限速步骤.本文重新设计了巨大芽孢杆菌P450 BM3的e TPs,大幅提高了其催化性能.通过在P450 BM3的e TP中引入芳香族氨基酸,“最佳”变体P2H02(A399Y/Q403F)的催化效率比P450 BM3野生型催化效率提高了12.9倍(kcat/KM从65.8 L mol^(-1)s^(-1)提高到913.5 L mol^(-1)s^(-1)).分子动力学模拟和电子传递分析表明,在辅因子FMN和血红素之间引入的芳香族氨基酸可以显著提高电子转移速率和酶催化性能.同时,在电子传递途径中引入酪氨酸可以保护P450的催化中心,使其避免被氧化性中间产物所破坏,从而提高其催化效率.此外,引入芳香族氨基酸的策略被证明对其他P450(如CYP116B3)同样有效,改造后的酶表现出显著提高的催化效率.综上,本文策略有望拓展到其他带有长程电子传递链的氧化还原酶上,成为改造的通用策略.Cytochrome P450s are versatile catalysts for biosynthesis applications.In the P450 catalytic cycle,two electrons are required to reduce the heme iron and activate the subsequent reductions through proposed electron transfer pathways(eTPs),which often represent the rate-limiting step in reactions.Herein,the P450 BM3 from Bacillus megaterium was engineered for improved catalytic performance by redesigning proposed eTPs.By introducing aromatic amino acids on eTPs of P450 BM3,the“best”variant P2H02(A399Y/Q403F)showed 13.9-fold improved catalytic efficiency(k_(cat)/K_(M)=913.5 L mol^(-1)s^(-1))compared with P450 BM3 WT(kcat/KM=65.8 L mol^(-1)s^(-1)).Molecular dynamics simulations and electron hopping pathways analysis revealed that aromatic amino acid substitutions bridging the cofactor flavin mononucleotide and heme iron could increase electron transfer rates and improve catalytic performance.Moreover,the introduction of tyrosines showed positive effects on catalytic efficiency by potentially protecting P450 from oxidative damage.In essence,engineering of eTPs by aromatic amino acid substitutions represents a powerful approach to design catalytically efficient P450s(such as CYP116B3)and could be expanded to other oxidoreductases relying on long-range electron transfer pathways.

关 键 词：定向进化 P450 BM3 蛋白质工程 电子传递 理性设计 

分 类 号：TQ426[化学工程]