Cytochrome P450 Enzyme Design by Constraining the Catalytic Pocket in a Diffusion Model
Qian Wang,
Xiaonan Liu,
Hejian Zhang,
Huanyu Chu,
Chao Shi,
Lei Zhang,
Jie Bai,
Pi Liu,
Jing Li,
Xiaoxi Zhu,
Yuwan Liu,
Zhangxin Chen,
Rong Huang,
Hong Chang,
Tian Liu,
Zhenzhan Chang,
Jian Cheng,
Huifeng Jiang
Affiliations
Qian Wang
Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, China.
Xiaonan Liu
Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, China.
Hejian Zhang
Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, China.
Huanyu Chu
Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, China.
Chao Shi
Department of Biochemistry and Biophysics, School of Basic Medical Sciences,
Peking University, Beijing 100191, China.
Lei Zhang
Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, China.
Jie Bai
Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, China.
Pi Liu
Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, China.
Jing Li
Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, China.
Xiaoxi Zhu
Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, China.
Yuwan Liu
Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, China.
Zhangxin Chen
Department of Biochemistry and Biophysics, School of Basic Medical Sciences,
Peking University, Beijing 100191, China.
Rong Huang
Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, China.
Hong Chang
Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, China.
Tian Liu
Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, China.
Zhenzhan Chang
Department of Biochemistry and Biophysics, School of Basic Medical Sciences,
Peking University, Beijing 100191, China.
Jian Cheng
Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, China.
Huifeng Jiang
Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, China.
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.
