Heterologous Overexpression of Cytochrome P450BM3 from <i>Bacillus megaterium</i> and Its Role in Gossypol Reduction
Gossypol is a polyphenolic toxic compound present in cotton plants. To determine whether the candidate cytochrome P450BM3 enzymes could reduce gossypol in vitro, functional recombinant cytochrome P450BM3 enzymes were successfully expressed in <i>E. coli</i>. Site-directed mutagenesis gen...
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2025-05-01
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| author | Wenpeng Fan Jingjing Cui Tongxiang Xu Shiheng Xu Zulibina Ainiwaer Qiyu Luo Caidie Wang |
| author_facet | Wenpeng Fan Jingjing Cui Tongxiang Xu Shiheng Xu Zulibina Ainiwaer Qiyu Luo Caidie Wang |
| author_sort | Wenpeng Fan |
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| description | Gossypol is a polyphenolic toxic compound present in cotton plants. To determine whether the candidate cytochrome P450BM3 enzymes could reduce gossypol in vitro, functional recombinant cytochrome P450BM3 enzymes were successfully expressed in <i>E. coli</i>. Site-directed mutagenesis generated mutants (R162H, R162K, Q129H, Q129N) to explore structural determinants of catalytic efficiency. Both wild-type P450BM3 and mutants exhibited significant ability to reduce gossypol levels, with R162H and R162K showing 33.4% and 24.2% reduced catalytic efficiency compared with the wild-type enzyme, respectively. Q129H and Q129N mutants maintained comparable catalytic efficiency to the wild type. Metabolomic profiling revealed two distinct reducing pathways catalyzed by wild-type P450BM3 and its mutants (R162H/Q129H), involving decarboxylation, hydroxylation, and C-C bond cleavage. This study demonstrated the feasibility of P450BM3 as a highly efficient biocatalyst for reducing gossypol levels, speculated that Arg162 might be a critical active residue, and hypothesized the potential pathways by which P450BM3 catalyzes the reduction of gossypol content, thereby providing a theoretical foundation for the enzymatic reduction of gossypol. |
| format | Article |
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| language | English |
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| spelling | doaj-art-cbca998ac9df4c6496b87456880acb5d2025-08-20T03:12:09ZengMDPI AGToxins2072-66512025-05-0117525310.3390/toxins17050253Heterologous Overexpression of Cytochrome P450BM3 from <i>Bacillus megaterium</i> and Its Role in Gossypol ReductionWenpeng Fan0Jingjing Cui1Tongxiang Xu2Shiheng Xu3Zulibina Ainiwaer4Qiyu Luo5Caidie Wang6Xinjiang Key Laboratory of Herbivore Nutrition for Meat & Milk, Research Center for Biofeed and Animal Gut Health, College of Animal Sciences, Xinjiang Agricultural University, Urumqi 830052, ChinaXinjiang Key Laboratory of Herbivore Nutrition for Meat & Milk, Research Center for Biofeed and Animal Gut Health, College of Animal Sciences, Xinjiang Agricultural University, Urumqi 830052, ChinaXinjiang Key Laboratory of Herbivore Nutrition for Meat & Milk, Research Center for Biofeed and Animal Gut Health, College of Animal Sciences, Xinjiang Agricultural University, Urumqi 830052, ChinaXinjiang Key Laboratory of Herbivore Nutrition for Meat & Milk, Research Center for Biofeed and Animal Gut Health, College of Animal Sciences, Xinjiang Agricultural University, Urumqi 830052, ChinaXinjiang Key Laboratory of Herbivore Nutrition for Meat & Milk, Research Center for Biofeed and Animal Gut Health, College of Animal Sciences, Xinjiang Agricultural University, Urumqi 830052, ChinaXinjiang Key Laboratory of Herbivore Nutrition for Meat & Milk, Research Center for Biofeed and Animal Gut Health, College of Animal Sciences, Xinjiang Agricultural University, Urumqi 830052, ChinaXinjiang Key Laboratory of Herbivore Nutrition for Meat & Milk, Research Center for Biofeed and Animal Gut Health, College of Animal Sciences, Xinjiang Agricultural University, Urumqi 830052, ChinaGossypol is a polyphenolic toxic compound present in cotton plants. To determine whether the candidate cytochrome P450BM3 enzymes could reduce gossypol in vitro, functional recombinant cytochrome P450BM3 enzymes were successfully expressed in <i>E. coli</i>. Site-directed mutagenesis generated mutants (R162H, R162K, Q129H, Q129N) to explore structural determinants of catalytic efficiency. Both wild-type P450BM3 and mutants exhibited significant ability to reduce gossypol levels, with R162H and R162K showing 33.4% and 24.2% reduced catalytic efficiency compared with the wild-type enzyme, respectively. Q129H and Q129N mutants maintained comparable catalytic efficiency to the wild type. Metabolomic profiling revealed two distinct reducing pathways catalyzed by wild-type P450BM3 and its mutants (R162H/Q129H), involving decarboxylation, hydroxylation, and C-C bond cleavage. This study demonstrated the feasibility of P450BM3 as a highly efficient biocatalyst for reducing gossypol levels, speculated that Arg162 might be a critical active residue, and hypothesized the potential pathways by which P450BM3 catalyzes the reduction of gossypol content, thereby providing a theoretical foundation for the enzymatic reduction of gossypol.https://www.mdpi.com/2072-6651/17/5/253cytochrome P450BM3heterologous overexpressiongossypolreduction pathways |
| spellingShingle | Wenpeng Fan Jingjing Cui Tongxiang Xu Shiheng Xu Zulibina Ainiwaer Qiyu Luo Caidie Wang Heterologous Overexpression of Cytochrome P450BM3 from <i>Bacillus megaterium</i> and Its Role in Gossypol Reduction Toxins cytochrome P450BM3 heterologous overexpression gossypol reduction pathways |
| title | Heterologous Overexpression of Cytochrome P450BM3 from <i>Bacillus megaterium</i> and Its Role in Gossypol Reduction |
| title_full | Heterologous Overexpression of Cytochrome P450BM3 from <i>Bacillus megaterium</i> and Its Role in Gossypol Reduction |
| title_fullStr | Heterologous Overexpression of Cytochrome P450BM3 from <i>Bacillus megaterium</i> and Its Role in Gossypol Reduction |
| title_full_unstemmed | Heterologous Overexpression of Cytochrome P450BM3 from <i>Bacillus megaterium</i> and Its Role in Gossypol Reduction |
| title_short | Heterologous Overexpression of Cytochrome P450BM3 from <i>Bacillus megaterium</i> and Its Role in Gossypol Reduction |
| title_sort | heterologous overexpression of cytochrome p450bm3 from i bacillus megaterium i and its role in gossypol reduction |
| topic | cytochrome P450BM3 heterologous overexpression gossypol reduction pathways |
| url | https://www.mdpi.com/2072-6651/17/5/253 |
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