Structure-guided manipulation of the regioselectivity of the cyclosporine A hydroxylase CYP-sb21 from Sebekia benihana
The cytochrome P450 enzyme CYP-sb21 from the rare actinomycete Sebekia benihana is capable of hydroxylating the immunosuppressive drug molecule cyclosporine A (CsA) primarily at the 4th N-methyl leucine (MeLeu4), giving rise to γ-hydroxy-N-methyl-l-Leu4-CsA (CsA-4-OH). This oxidative modification of...
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KeAi Communications Co., Ltd.
2020-09-01
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| Series: | Synthetic and Systems Biotechnology |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405805X20300491 |
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| author | Fengwei Li Li Ma Xingwang Zhang Jingfei Chen Feifei Qi Yinyue Huang Zepeng Qu Lishan Yao Wei Zhang Eung-Soo Kim Shengying Li |
| author_facet | Fengwei Li Li Ma Xingwang Zhang Jingfei Chen Feifei Qi Yinyue Huang Zepeng Qu Lishan Yao Wei Zhang Eung-Soo Kim Shengying Li |
| author_sort | Fengwei Li |
| collection | DOAJ |
| description | The cytochrome P450 enzyme CYP-sb21 from the rare actinomycete Sebekia benihana is capable of hydroxylating the immunosuppressive drug molecule cyclosporine A (CsA) primarily at the 4th N-methyl leucine (MeLeu4), giving rise to γ-hydroxy-N-methyl-l-Leu4-CsA (CsA-4-OH). This oxidative modification of CsA leads to dramatically reduced immunosuppressive activity while retaining the hair growth-promoting side-effect, thus demonstrating great application potential in both pharmaceutical and cosmetic industries. However, this P450 enzyme also hydroxylates CsA at the unwanted position of the 9th N-methyl leucine (MeLeu9), indicating that the regioselectivity needs to be improved for the development of CsA-4-OH into a commercial hair growth stimulator. Herein, we report the crystal structure of CYP-sb21 in its substrate-free form at 1.85 Å. Together with sequence and 3D structure comparisons, Autodock-based substrate docking, molecular dynamics (MD) simulation, and site-directed mutagenesis, we identified a number of key residues including R294, E264, and M179 that can improve catalytic efficiency or change the regioselectivity of CYP-sb21 towards CsA, setting the stage for better enzymatic preparation of CsA-4-OH. This study also provides new insights into the substrate recognition and binding mechanism of P450 enzymes that accommodate bulky substrates. |
| first_indexed | 2024-04-24T08:17:41Z |
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| id | doaj.art-ebd52a13a250486da80b3d3725a9bc0e |
| institution | Directory Open Access Journal |
| issn | 2405-805X |
| language | English |
| last_indexed | 2024-04-24T08:17:41Z |
| publishDate | 2020-09-01 |
| publisher | KeAi Communications Co., Ltd. |
| record_format | Article |
| series | Synthetic and Systems Biotechnology |
| spelling | doaj.art-ebd52a13a250486da80b3d3725a9bc0e2024-04-17T03:05:42ZengKeAi Communications Co., Ltd.Synthetic and Systems Biotechnology2405-805X2020-09-0153236243Structure-guided manipulation of the regioselectivity of the cyclosporine A hydroxylase CYP-sb21 from Sebekia benihanaFengwei Li0Li Ma1Xingwang Zhang2Jingfei Chen3Feifei Qi4Yinyue Huang5Zepeng Qu6Lishan Yao7Wei Zhang8Eung-Soo Kim9Shengying Li10State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, China; Shandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101, China; Corresponding author. State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, China.State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, ChinaState Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, ChinaShandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101, ChinaShandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101, ChinaState Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, ChinaShandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101, ChinaShandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101, ChinaState Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, China; Shandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101, ChinaDepartment of Biological Engineering, Inha University, Incheon, 22212, South Korea; Corresponding author.State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, China; Shandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shandong, 266237, China; Corresponding author. State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, China.The cytochrome P450 enzyme CYP-sb21 from the rare actinomycete Sebekia benihana is capable of hydroxylating the immunosuppressive drug molecule cyclosporine A (CsA) primarily at the 4th N-methyl leucine (MeLeu4), giving rise to γ-hydroxy-N-methyl-l-Leu4-CsA (CsA-4-OH). This oxidative modification of CsA leads to dramatically reduced immunosuppressive activity while retaining the hair growth-promoting side-effect, thus demonstrating great application potential in both pharmaceutical and cosmetic industries. However, this P450 enzyme also hydroxylates CsA at the unwanted position of the 9th N-methyl leucine (MeLeu9), indicating that the regioselectivity needs to be improved for the development of CsA-4-OH into a commercial hair growth stimulator. Herein, we report the crystal structure of CYP-sb21 in its substrate-free form at 1.85 Å. Together with sequence and 3D structure comparisons, Autodock-based substrate docking, molecular dynamics (MD) simulation, and site-directed mutagenesis, we identified a number of key residues including R294, E264, and M179 that can improve catalytic efficiency or change the regioselectivity of CYP-sb21 towards CsA, setting the stage for better enzymatic preparation of CsA-4-OH. This study also provides new insights into the substrate recognition and binding mechanism of P450 enzymes that accommodate bulky substrates.http://www.sciencedirect.com/science/article/pii/S2405805X20300491Cytochrome P450 monooxygenaseCrystal structureCyclosporine ARegioselectivityHair growth stimulator |
| spellingShingle | Fengwei Li Li Ma Xingwang Zhang Jingfei Chen Feifei Qi Yinyue Huang Zepeng Qu Lishan Yao Wei Zhang Eung-Soo Kim Shengying Li Structure-guided manipulation of the regioselectivity of the cyclosporine A hydroxylase CYP-sb21 from Sebekia benihana Synthetic and Systems Biotechnology Cytochrome P450 monooxygenase Crystal structure Cyclosporine A Regioselectivity Hair growth stimulator |
| title | Structure-guided manipulation of the regioselectivity of the cyclosporine A hydroxylase CYP-sb21 from Sebekia benihana |
| title_full | Structure-guided manipulation of the regioselectivity of the cyclosporine A hydroxylase CYP-sb21 from Sebekia benihana |
| title_fullStr | Structure-guided manipulation of the regioselectivity of the cyclosporine A hydroxylase CYP-sb21 from Sebekia benihana |
| title_full_unstemmed | Structure-guided manipulation of the regioselectivity of the cyclosporine A hydroxylase CYP-sb21 from Sebekia benihana |
| title_short | Structure-guided manipulation of the regioselectivity of the cyclosporine A hydroxylase CYP-sb21 from Sebekia benihana |
| title_sort | structure guided manipulation of the regioselectivity of the cyclosporine a hydroxylase cyp sb21 from sebekia benihana |
| topic | Cytochrome P450 monooxygenase Crystal structure Cyclosporine A Regioselectivity Hair growth stimulator |
| url | http://www.sciencedirect.com/science/article/pii/S2405805X20300491 |
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