Regulating the N-oxidation selectivity of P450BM3 monooxygenases for N-heterocycles through computer-assisted structure-guided design
Abstract N-oxidation of N-heterocycles is essential in the synthesis of natural products but challenging due to low efficacy and poor regioselectivity. In this study, the N-oxidation selective potential of P450BM3 from Bacillus megaterium for N-heterocyclic compounds is investigated. Here, twelve am...
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Nature Portfolio
2025-07-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-61773-3 |
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| author | Liu Yang Zhongji Pu Jianping Wu Xiaofeng Liu Zhe Wang Haoran Yu Liuwei Wang Yan Meng Gang Xu Lirong Yang Wenlong Zheng |
| author_facet | Liu Yang Zhongji Pu Jianping Wu Xiaofeng Liu Zhe Wang Haoran Yu Liuwei Wang Yan Meng Gang Xu Lirong Yang Wenlong Zheng |
| author_sort | Liu Yang |
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| description | Abstract N-oxidation of N-heterocycles is essential in the synthesis of natural products but challenging due to low efficacy and poor regioselectivity. In this study, the N-oxidation selective potential of P450BM3 from Bacillus megaterium for N-heterocyclic compounds is investigated. Here, twelve amino acids located in the active center, including A74, L75, V78, A82, F87, I263, A264, A328, P329, A330, I401, and L437, are investigated by site-saturation mutation. As a result, F87, A264, L75, V78, A328, I401, and L437 are identified as hotspot residues. Subsequently, the combinatorial active-site saturation test/iterative saturation mutagenesis strategy is performed. Using quinoline as a model substrate, the mutant F87G/A264G/A328L exhibits N-oxidation selectivity of up to 99.0%, with a conversion rate of 99.3%. Molecular dynamics simulations uncover a “push-pull” molecular mechanism elucidating the pivotal role of steric factors in determining substrate recognition and N-oxidation selectivity. This study provides an efficient N-oxide synthesis method and insights into P450BM3’s molecular mechanisms. |
| format | Article |
| id | doaj-art-e8979ffcfab74b6c9321c6a7594a9188 |
| institution | DOAJ |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
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| series | Nature Communications |
| spelling | doaj-art-e8979ffcfab74b6c9321c6a7594a91882025-08-20T03:05:14ZengNature PortfolioNature Communications2041-17232025-07-0116111210.1038/s41467-025-61773-3Regulating the N-oxidation selectivity of P450BM3 monooxygenases for N-heterocycles through computer-assisted structure-guided designLiu Yang0Zhongji Pu1Jianping Wu2Xiaofeng Liu3Zhe Wang4Haoran Yu5Liuwei Wang6Yan Meng7Gang Xu8Lirong Yang9Wenlong Zheng10Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang UniversityXianghu laboratoryInstitute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang UniversityShaoxing Institute, Zhejiang UniversityInstitute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang UniversityInstitute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang UniversityInstitute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang UniversityInstitute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang UniversityInstitute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang UniversityInstitute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang UniversityInstitute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang UniversityAbstract N-oxidation of N-heterocycles is essential in the synthesis of natural products but challenging due to low efficacy and poor regioselectivity. In this study, the N-oxidation selective potential of P450BM3 from Bacillus megaterium for N-heterocyclic compounds is investigated. Here, twelve amino acids located in the active center, including A74, L75, V78, A82, F87, I263, A264, A328, P329, A330, I401, and L437, are investigated by site-saturation mutation. As a result, F87, A264, L75, V78, A328, I401, and L437 are identified as hotspot residues. Subsequently, the combinatorial active-site saturation test/iterative saturation mutagenesis strategy is performed. Using quinoline as a model substrate, the mutant F87G/A264G/A328L exhibits N-oxidation selectivity of up to 99.0%, with a conversion rate of 99.3%. Molecular dynamics simulations uncover a “push-pull” molecular mechanism elucidating the pivotal role of steric factors in determining substrate recognition and N-oxidation selectivity. This study provides an efficient N-oxide synthesis method and insights into P450BM3’s molecular mechanisms.https://doi.org/10.1038/s41467-025-61773-3 |
| spellingShingle | Liu Yang Zhongji Pu Jianping Wu Xiaofeng Liu Zhe Wang Haoran Yu Liuwei Wang Yan Meng Gang Xu Lirong Yang Wenlong Zheng Regulating the N-oxidation selectivity of P450BM3 monooxygenases for N-heterocycles through computer-assisted structure-guided design Nature Communications |
| title | Regulating the N-oxidation selectivity of P450BM3 monooxygenases for N-heterocycles through computer-assisted structure-guided design |
| title_full | Regulating the N-oxidation selectivity of P450BM3 monooxygenases for N-heterocycles through computer-assisted structure-guided design |
| title_fullStr | Regulating the N-oxidation selectivity of P450BM3 monooxygenases for N-heterocycles through computer-assisted structure-guided design |
| title_full_unstemmed | Regulating the N-oxidation selectivity of P450BM3 monooxygenases for N-heterocycles through computer-assisted structure-guided design |
| title_short | Regulating the N-oxidation selectivity of P450BM3 monooxygenases for N-heterocycles through computer-assisted structure-guided design |
| title_sort | regulating the n oxidation selectivity of p450bm3 monooxygenases for n heterocycles through computer assisted structure guided design |
| url | https://doi.org/10.1038/s41467-025-61773-3 |
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