Engineering Cytochrome P450BM3 Enzymes for Direct Nitration of Unsaturated Hydrocarbons
Xiling Wang
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
These authors contributed equally to this work.
Contribution: Data curation (lead), Funding acquisition (equal), Investigation (lead), Methodology (lead), Writing - original draft (equal), Writing - review & editing (equal)
Search for more papers by this authorXiaodan Lin
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
University of Chinese Academy of Sciences, Beijing, 100049 China
These authors contributed equally to this work.
Contribution: Data curation (equal), Investigation (equal), Methodology (equal), Writing - original draft (supporting), Writing - review & editing (supporting)
Search for more papers by this authorYiping Jiang
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
These authors contributed equally to this work.
Contribution: Data curation (lead), Funding acquisition (equal), Investigation (lead), Methodology (lead), Writing - original draft (equal), Writing - review & editing (equal)
Search for more papers by this authorXiangquan Qin
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
Department of Chemistry, Yanbian University Yanji, Jilin, 133002 China
These authors contributed equally to this work.
Contribution: Data curation (equal), Investigation (supporting), Methodology (supporting), Writing - original draft (supporting), Writing - review & editing (supporting)
Search for more papers by this authorNana Ma
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
University of Chinese Academy of Sciences, Beijing, 100049 China
Contribution: Data curation (supporting), Investigation (supporting), Methodology (supporting)
Search for more papers by this authorFuquan Yao
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
Contribution: Data curation (supporting), Investigation (supporting)
Search for more papers by this authorSheng Dong
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
Contribution: Data curation (supporting), Investigation (supporting)
Search for more papers by this authorChuanfei Liu
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
Contribution: Data curation (supporting)
Search for more papers by this authorYingang Feng
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
Contribution: Methodology (supporting), Supervision (supporting), Writing - review & editing (supporting)
Search for more papers by this authorLongyi Jin
Department of Chemistry, Yanbian University Yanji, Jilin, 133002 China
Contribution: Methodology (supporting), Supervision (supporting), Writing - review & editing (supporting)
Search for more papers by this authorMo Xian
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
Contribution: Methodology (supporting), Writing - review & editing (supporting)
Search for more papers by this authorCorresponding Author
Zhiqi Cong
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
University of Chinese Academy of Sciences, Beijing, 100049 China
Contribution: Conceptualization (lead), Formal analysis (lead), Funding acquisition (lead), Investigation (lead), Methodology (lead), Project administration (lead), Supervision (lead), Writing - original draft (lead), Writing - review & editing (lead)
Search for more papers by this authorXiling Wang
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
These authors contributed equally to this work.
Contribution: Data curation (lead), Funding acquisition (equal), Investigation (lead), Methodology (lead), Writing - original draft (equal), Writing - review & editing (equal)
Search for more papers by this authorXiaodan Lin
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
University of Chinese Academy of Sciences, Beijing, 100049 China
These authors contributed equally to this work.
Contribution: Data curation (equal), Investigation (equal), Methodology (equal), Writing - original draft (supporting), Writing - review & editing (supporting)
Search for more papers by this authorYiping Jiang
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
These authors contributed equally to this work.
Contribution: Data curation (lead), Funding acquisition (equal), Investigation (lead), Methodology (lead), Writing - original draft (equal), Writing - review & editing (equal)
Search for more papers by this authorXiangquan Qin
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
Department of Chemistry, Yanbian University Yanji, Jilin, 133002 China
These authors contributed equally to this work.
Contribution: Data curation (equal), Investigation (supporting), Methodology (supporting), Writing - original draft (supporting), Writing - review & editing (supporting)
Search for more papers by this authorNana Ma
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
University of Chinese Academy of Sciences, Beijing, 100049 China
Contribution: Data curation (supporting), Investigation (supporting), Methodology (supporting)
Search for more papers by this authorFuquan Yao
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
Contribution: Data curation (supporting), Investigation (supporting)
Search for more papers by this authorSheng Dong
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
Contribution: Data curation (supporting), Investigation (supporting)
Search for more papers by this authorChuanfei Liu
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
Contribution: Data curation (supporting)
Search for more papers by this authorYingang Feng
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
Contribution: Methodology (supporting), Supervision (supporting), Writing - review & editing (supporting)
Search for more papers by this authorLongyi Jin
Department of Chemistry, Yanbian University Yanji, Jilin, 133002 China
Contribution: Methodology (supporting), Supervision (supporting), Writing - review & editing (supporting)
Search for more papers by this authorMo Xian
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
Contribution: Methodology (supporting), Writing - review & editing (supporting)
Search for more papers by this authorCorresponding Author
Zhiqi Cong
CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China
University of Chinese Academy of Sciences, Beijing, 100049 China
Contribution: Conceptualization (lead), Formal analysis (lead), Funding acquisition (lead), Investigation (lead), Methodology (lead), Project administration (lead), Supervision (lead), Writing - original draft (lead), Writing - review & editing (lead)
Search for more papers by this authorAbstract
Applications of the peroxidase activity of cytochrome P450 enzymes in synthetic chemistry remain largely unexplored. We present herein a protein engineering strategy to increase cytochrome P450BM3 peroxidase activity for the direct nitration of aromatic compounds and terminal aryl-substituted olefins in the presence of a dual-functional small molecule (DFSM). Site-directed mutations of key active-site residues allowed the efficient regulation of steric effects to limit substrate access and, thus, a significant decrease in monooxygenation activity and increase in peroxidase activity. Nitration of several phenol and aniline compounds also yielded ortho- and para-nitration products with moderate-to-high total turnover numbers. Besides direct aromatic nitration by P450 variants using nitrite as a nitrating agent, we also demonstrated the use of the DFSM-facilitated P450 peroxidase system for the nitration of the vinyl group of styrene and its derivatives.
Conflict of interest
The authors declare no conflict of interest.
Open Research
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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