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 ...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.展开更多
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)+3 种基金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).
文摘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.
