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

作　　者：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 

机构地区：[1]Key Laboratory of Engineering Biology for Low-Carbon Manufacturing,Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China [2]University of Chinese Academy of Sciences,Beijing 100049,China [3]National Center of Technology Innovation for Synthetic Biology,Tianjin 300308,China [4]College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China [5]Department of Biochemistry and Biophysics,School of Basic Medical Sciences,Peking University,Beijing 100191,China [6]College of Life Science and Technology,Wuhan Polytechnic University,Wuhan,Hubei 430023,China [7]State Key Laboratory of Elemento-Organic Chemistry,College of Chemistry,Nankai University,Tianjin 300071,China [8]College of Life Science,Nankai University,Tianjin 300071,China

出　　处：《Research》2025年第1期618-630,共13页研究(英文)

基　　金：funding from the National Key R&D Program of China(grant no.2019YFA0905700 and 2021YFC2103500);the National Natural Science Foundation of China(no.32371499);the China Postdoctoral Science Foundation(grant no.2019M661032);the National Natural Science Foundation of China(NSFC,grant nos.31901026 and 32171418);the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(nos.TSBICIP-KJGG-002-02 and TSBICIP-CXRC-015);the Tianjin Science Fund for Distinguished Young Scholars(no.18JCJQJC48300).

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.

关 键 词：cytochrome P progressive forward accumulationwe cytochrome p enzymes molecular mechanism ancestral sequence reconstructionreverse mutation assayand de novo design catalytic pocket functional innovation 

分 类 号：Q55[生物学—生物化学]