Cytochrome P450 Enzyme Design by Constraining the Catalytic Pocket in a Diffusion Model

被引:2
|
作者
Wang, Qian [1 ,2 ,3 ]
Liu, Xiaonan [1 ,2 ,3 ]
Zhang, Hejian [1 ,3 ,4 ]
Chu, Huanyu [1 ,2 ]
Shi, Chao [5 ]
Zhang, Lei [1 ,6 ]
Bai, Jie [1 ,3 ]
Liu, Pi [1 ,3 ]
Li, Jing [1 ,3 ,7 ,8 ]
Zhu, Xiaoxi [1 ,2 ,3 ]
Liu, Yuwan [1 ,3 ]
Chen, Zhangxin [5 ]
Huang, Rong [1 ,3 ]
Chang, Hong [1 ,3 ]
Liu, Tian [1 ,3 ]
Chang, Zhenzhan [5 ]
Cheng, Jian [1 ,3 ]
Jiang, Huifeng [1 ,3 ]
机构
[1] Chinese Acad Sci, Tianjin Inst Ind Biotechnol, Key Lab Engn Biol Low Carbon Mfg, Tianjin 300308, Peoples R China
[2] Univ Chinese Acad Sci, Beijing 100049, Peoples R China
[3] Natl Ctr Technol Innovat Synthet Biol, Tianjin 300308, Peoples R China
[4] Tianjin Univ Sci & Technol, Coll Biotechnol, Tianjin 300457, Peoples R China
[5] Peking Univ, Sch Basic Med Sci, Dept Biochem & Biophys, Beijing 100191, Peoples R China
[6] Wuhan Polytech Univ, Coll Life Sci & Technol, Wuhan 430023, Peoples R China
[7] Nankai Univ, Coll Chem, State Key Lab Elemento Organ Chem, Tianjin 300071, Peoples R China
[8] Nankai Univ, Coll Life Sci, Tianjin 300071, Peoples R China
基金
中国博士后科学基金; 国家重点研发计划; 中国国家自然科学基金;
关键词
PROTEIN-SEQUENCE DESIGN; MOLECULAR-DYNAMICS; DIRECTED EVOLUTION; GENES; PREDICTION; ORIGIN; P450; REACTIVITY; MECHANISM; INSIGHTS;
D O I
10.34133/research.0413
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Although cytochrome P450 enzymes are the most versatile biocatalysts in nature, there is insufficient comprehension of the molecular mechanism underlying their functional innovation process. Here, by combining ancestral sequence reconstruction, reverse mutation assay, and progressive forward accumulation, we identified 5 founder residues in the catalytic pocket of flavone 6-hydroxylase (F6H) and proposed a "3-point fixation" model to elucidate the functional innovation mechanisms of P450s in nature. According to this design principle of catalytic pocket, we further developed a de novo diffusion model (P450Diffusion) to generate artificial P450s. Ultimately, among the 17 non-natural P450s we generated, 10 designs exhibited significant F6H activity and 6 exhibited a 1.3- to 3.5-fold increase in catalytic capacity compared to the natural CYP706X1. This work not only explores the design principle of catalytic pockets of P450s, but also provides an insight into the artificial design of P450 enzymes with desired functions.
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页数:13
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