Synthetic Biology Journal ›› 2022, Vol. 3 ›› Issue (3): 587-601.DOI: 10.12211/2096-8280.2021-053

• Research Article • Previous Articles     Next Articles

Studies on the functional modulating effect of redox partners on the cytochrome P450 enzyme MycG

Chaofan YANG, Yuchao JIANG, Moli SANG, Shengying LI, Wei ZHANG   

  1. Microbial Technology Institute,State key Laboratory of Microbial Technology,Shandong University,Qingdao 266237,Shandong,China
  • Received:2021-04-30 Revised:2021-06-08 Online:2022-07-13 Published:2022-06-30
  • Contact: Wei ZHANG

还原伴侣对细胞色素P450酶MycG功能调控的研究

杨超凡, 姜玉超, 桑茉莉, 李盛英, 张伟   

  1. 山东大学微生物技术研究院,山东大学微生物技术国家重点实验室,山东 青岛 266237
  • 通讯作者: 张伟
  • 作者简介:杨超凡(1997—),女,硕士研究生。主要研究方向为酶工程、合成生物学。 E-mail:yangchaofan@mail.sdu.edu.cn
    张伟(1982—),男,博士,教授。主要研究方向为合成生物学、天然产物生物合成、酶工程。 E-mail:zhang_wei@sdu.edu.cn
  • 基金资助:
    国家重点研发计划(2019YFA0905700);国家自然科学基金(82022066)

Abstract:

Cytochrome P450 enzymes, which are widely involved in the key biosynthetic steps of many natural products, are considered as the most versatile biocatalysts in the nature. Redox partners responsible for electron transfer are indispensable for most P450 catalytic reactions. During the long evolution process, various P450-redox partner systems have been constituted by protein fusion and recombination. The protein-protein interaction and recognition between P450 and redox partner(s) are not only the key factor for functional reconstitution of P450s, but also can affect and change the catalytic functions and properties of P450 enzymes. To address the issue that how the choice and recombination of different redox partners affect the catalytic behaviors (reaction type, catalytic efficiency, product distribution, and electron transport) of the P450 enzyme, a number of fusion and separation P450 systems were constructed based on the two-domain redox partner protein RhFRED and P450 MycG. Specifically, the two domains of RhFRED were separated and expressed as stand-alone FMN and Fe2S2 proteins. The positions of these two domains and MycG were shuffled and engineered. In vitro biochemical reactions were carried out to explore the effect of different redox partner combinations on the MycG-catalyzed bioconversion of mycinamicin-Ⅳ (M-Ⅳ) and the electron transfer efficiency as well. As results, among the 20 tested catalytic systems, the functions of the multifunctional P450 enzyme MycG were successfully reconstituted through 12 combinations, in which three oxidative products including M-Ⅰ, M-Ⅱ and M-Ⅴ, and one demethylation product dMe-M-Ⅳ were produced. Besides, 4 combinations only led to the production of three oxidative products. In addition, no products were detected in the other 4 combinations. By simulating natural evolutionary strategy, the effects of three different catalytic systems of redox partner(s) and P450 enzymes, together with different protein organization forms on the functional modulating of MycG, were studied systematically. These results indicate that the P450 catalytic properties are affected and modulated by not only the identity, but also the position and organization of redox partner (s), probably via alternative protein-protein interactions.

Key words: cytochrome P450 enzymes, redox partners, MycG, RhFRED, redox partner engineering

摘要:

细胞色素P450酶负责催化许多天然产物生物合成过程中的关键反应,是自然界中最具催化多样性的生物催化剂。还原伴侣蛋白对P450催化功能至关重要,在进化过程中两者之间通过融合或分离方式演化出多种不同的催化系统。还原伴侣与P450酶的适配性往往是造成P450酶功能重建失败或低效的关键因素,也能够影响和改变P450酶的催化功能和性质。不同还原伴侣与P450酶的组合与互作如何影响P450酶的催化功能、催化效率和产物分布是值得深入探究的科学问题。本研究以还原伴侣蛋白RhFRED和P450酶MycG为研究对象,通过构建不同的融合和分离蛋白组合,系统研究了还原伴侣与P450酶的适配和相互作用模式对P450酶催化功能的影响。将RhFRED两个结构功能域拆解为两个独立蛋白FMN和Fe2S2,与P450酶MycG三者之间利用还原伴侣工程构建分离或融合型蛋白,通过体外生化反应探究不同组合方式下MycG对底物麦新米星M-Ⅳ催化功能以及电子传递效率的影响。在构建的20个还原伴侣与P450酶的“非天然”催化组合中,16个组合能够成功重建MycG的催化功能,其中12个组合中MycG能够催化产生3种氧化产物麦新米星M-Ⅰ、M-Ⅱ和M-Ⅴ以及1种脱甲基产物dMe-M-Ⅳ;4个组合仅产生氧化产物M-Ⅰ、M-Ⅱ和M-Ⅴ;剩余4个组合未检测到任何反应产物。通过模拟自然进化,系统研究了还原伴侣与P450酶构成的3种不同催化系统(单、双、三组分)以及单组分和双组分系统中不同的蛋白组织形式对MycG催化功能和性质的影响,结果表明不同还原伴侣系统和组合/组织方式能够通过蛋白-蛋白相互作用显著影响P450的催化功能、催化效率和催化性质。

关键词: 细胞色素P450酶, 还原伴侣, MycG, RhFRED, 还原伴侣工程

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