A Novel Thermostable Cytochrome P450 from Sequence-Based Metagenomics of Binh Chau Hot Spring as a Promising Catalyst for Testosterone Conversion
Biotechnological applications of cytochromes P450 show difficulties, such as low activity, thermal and/or solvent instability, narrow substrate specificity and redox partner dependence. In an attempt to overcome these limitations, an exploitation of novel thermophilic P450 enzymes from nature via un...
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| Format: | Article |
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MDPI AG
2020-09-01
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| Series: | Catalysts |
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| Online Access: | https://www.mdpi.com/2073-4344/10/9/1083 |
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| author | Kim-Thoa Nguyen Ngọc-Lan Nguyen Nguyen Van Tung Huy Hoang Nguyen Mohammed Milhim Thi-Thanh-Xuan Le Thi-Hong-Nhung Lai Thi-Tuyet-Minh Phan Rita Bernhardt |
| author_facet | Kim-Thoa Nguyen Ngọc-Lan Nguyen Nguyen Van Tung Huy Hoang Nguyen Mohammed Milhim Thi-Thanh-Xuan Le Thi-Hong-Nhung Lai Thi-Tuyet-Minh Phan Rita Bernhardt |
| author_sort | Kim-Thoa Nguyen |
| collection | DOAJ |
| description | Biotechnological applications of cytochromes P450 show difficulties, such as low activity, thermal and/or solvent instability, narrow substrate specificity and redox partner dependence. In an attempt to overcome these limitations, an exploitation of novel thermophilic P450 enzymes from nature via uncultured approaches is desirable due to their great advantages that can resolve nearly all mentioned impediments. From the metagenomics library of the Binh Chau hot spring, an open reading frame (ORF) encoding a thermostable cytochrome P450—designated as P450-T3—which shared 66.6% amino acid sequence identity with CYP109C2 of <i>Sorangium cellulosum</i> So ce56 was selected for further identification and characterization. The ORF was synthesized artificially and heterologously expressed in <i>Escherichia coli</i> C43(DE3) using the pET17b system. The purified enzyme had a molecular weight of approximately 43 kDa. The melting temperature of the purified enzyme was 76.2 °C and its apparent half-life at 60 °C was 38.7 min. Redox partner screening revealed that P450-T3 was reduced well by the mammalian AdR-Adx<sub>4-108</sub> and the yeast Arh1-Etp1 redox partners. Lauric acid, palmitic acid, embelin, retinoic acid (<i>all-trans</i>) and retinoic acid (<i>13-cis</i>) demonstrated binding to P450-T3. Interestingly, P450-T3 also bound and converted testosterone. Overall, P450-T3 might become a good candidate for biocatalytic applications on a larger scale. |
| first_indexed | 2024-03-10T16:13:44Z |
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| id | doaj.art-9327a2036de9420db7482264a9f3e9c0 |
| institution | Directory Open Access Journal |
| issn | 2073-4344 |
| language | English |
| last_indexed | 2024-03-10T16:13:44Z |
| publishDate | 2020-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Catalysts |
| spelling | doaj.art-9327a2036de9420db7482264a9f3e9c02023-11-20T14:16:40ZengMDPI AGCatalysts2073-43442020-09-01109108310.3390/catal10091083A Novel Thermostable Cytochrome P450 from Sequence-Based Metagenomics of Binh Chau Hot Spring as a Promising Catalyst for Testosterone ConversionKim-Thoa Nguyen0Ngọc-Lan Nguyen1Nguyen Van Tung2Huy Hoang Nguyen3Mohammed Milhim4Thi-Thanh-Xuan Le5Thi-Hong-Nhung Lai6Thi-Tuyet-Minh Phan7Rita Bernhardt8Institute of Biotechnology, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 100000, VietnamGraduate University of Science and Technology, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 100000, VietnamGraduate University of Science and Technology, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 100000, VietnamGraduate University of Science and Technology, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 100000, VietnamDepartment of Biochemistry, Saarland University, 66123 Saarbrücken, GermanyInstitute of Biotechnology, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 100000, VietnamInstitute of Biotechnology, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 100000, VietnamInstitute of Biotechnology, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 100000, VietnamDepartment of Biochemistry, Saarland University, 66123 Saarbrücken, GermanyBiotechnological applications of cytochromes P450 show difficulties, such as low activity, thermal and/or solvent instability, narrow substrate specificity and redox partner dependence. In an attempt to overcome these limitations, an exploitation of novel thermophilic P450 enzymes from nature via uncultured approaches is desirable due to their great advantages that can resolve nearly all mentioned impediments. From the metagenomics library of the Binh Chau hot spring, an open reading frame (ORF) encoding a thermostable cytochrome P450—designated as P450-T3—which shared 66.6% amino acid sequence identity with CYP109C2 of <i>Sorangium cellulosum</i> So ce56 was selected for further identification and characterization. The ORF was synthesized artificially and heterologously expressed in <i>Escherichia coli</i> C43(DE3) using the pET17b system. The purified enzyme had a molecular weight of approximately 43 kDa. The melting temperature of the purified enzyme was 76.2 °C and its apparent half-life at 60 °C was 38.7 min. Redox partner screening revealed that P450-T3 was reduced well by the mammalian AdR-Adx<sub>4-108</sub> and the yeast Arh1-Etp1 redox partners. Lauric acid, palmitic acid, embelin, retinoic acid (<i>all-trans</i>) and retinoic acid (<i>13-cis</i>) demonstrated binding to P450-T3. Interestingly, P450-T3 also bound and converted testosterone. Overall, P450-T3 might become a good candidate for biocatalytic applications on a larger scale.https://www.mdpi.com/2073-4344/10/9/1083thermostable P450CYP109CmetagenomicP450-T3fatty acidstestosterone |
| spellingShingle | Kim-Thoa Nguyen Ngọc-Lan Nguyen Nguyen Van Tung Huy Hoang Nguyen Mohammed Milhim Thi-Thanh-Xuan Le Thi-Hong-Nhung Lai Thi-Tuyet-Minh Phan Rita Bernhardt A Novel Thermostable Cytochrome P450 from Sequence-Based Metagenomics of Binh Chau Hot Spring as a Promising Catalyst for Testosterone Conversion Catalysts thermostable P450 CYP109C metagenomic P450-T3 fatty acids testosterone |
| title | A Novel Thermostable Cytochrome P450 from Sequence-Based Metagenomics of Binh Chau Hot Spring as a Promising Catalyst for Testosterone Conversion |
| title_full | A Novel Thermostable Cytochrome P450 from Sequence-Based Metagenomics of Binh Chau Hot Spring as a Promising Catalyst for Testosterone Conversion |
| title_fullStr | A Novel Thermostable Cytochrome P450 from Sequence-Based Metagenomics of Binh Chau Hot Spring as a Promising Catalyst for Testosterone Conversion |
| title_full_unstemmed | A Novel Thermostable Cytochrome P450 from Sequence-Based Metagenomics of Binh Chau Hot Spring as a Promising Catalyst for Testosterone Conversion |
| title_short | A Novel Thermostable Cytochrome P450 from Sequence-Based Metagenomics of Binh Chau Hot Spring as a Promising Catalyst for Testosterone Conversion |
| title_sort | novel thermostable cytochrome p450 from sequence based metagenomics of binh chau hot spring as a promising catalyst for testosterone conversion |
| topic | thermostable P450 CYP109C metagenomic P450-T3 fatty acids testosterone |
| url | https://www.mdpi.com/2073-4344/10/9/1083 |
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