Zhan Liu, Jing Pang, Yi Li, Daijing Wei, Jing Yang, Xuefei Wang, Yinggang Luo
{"title":"参与单萜吲哚生物碱生物合成的细胞色素 P450 酶的催化选择性和进化","authors":"Zhan Liu, Jing Pang, Yi Li, Daijing Wei, Jing Yang, Xuefei Wang, Yinggang Luo","doi":"10.1111/ppl.14515","DOIUrl":null,"url":null,"abstract":"Cytochrome P450 enzyme (CYP)‐catalyzed functional group transformations are pivotal in the biosynthesis of metabolic intermediates and products, as exemplified by the CYP‐catalyzed C7‐hydroxylation and the subsequent C7‐C8 bond cleavage reaction responsible for the biosynthesis of the well‐known antitumor monoterpene indole alkaloid (MIA) camptothecin. To determine the key amino acid residues responsible for the catalytic selectivity of the CYPs involved in MIA biosynthesis, we characterized the enzymes CYP72A728 and CYP72A729 as stereoselective 7‐deoxyloganic acid 7‐hydroxylases (7DLHs). We then conducted a comparative analysis of the amino acid sequences and the predicted structures of the CYP72A homologs involved in camptothecin biosynthesis, as well as those of the CYP72A homologs implicated in the pharmaceutically significant MIAs biosynthesis in <jats:italic>Catharanthus roseus</jats:italic>. The crucial amino acid residues for the catalytic selectivity of the CYP72A‐catalyzed reactions were identified through fragmental and individual residue replacement, catalytic activity assays, molecular docking, and molecular dynamic simulations analysis. The fragments 1 and 3 of CYP72A565 were crucial for its C7‐hydroxylation and C7‐C8 bond cleavage activities. Mutating fragments 1 and 2 of CYP72A565 transformed the bifunctional CYP72A565 into a monofunctional 7DLH. Evolutionary analysis of the CYP72A homologs suggested that the bifunctional CYP72A in MIA‐producing plants may have evolved into a monofunctional CYP72A. The gene pairs <jats:italic>CYP72A728‐CYP72A610</jats:italic> and <jats:italic>CYP72A729‐CYP72A565</jats:italic> may have originated from a whole genome duplication event. This study provides a molecular basis for the CYP72A‐catalyzed hydroxylation and C‐C bond cleavage activities of CYP72A565, as well as evolutionary insights of CYP72A homologs involved in MIAs biosynthesis.","PeriodicalId":20164,"journal":{"name":"Physiologia plantarum","volume":null,"pages":null},"PeriodicalIF":5.4000,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Catalytic selectivity and evolution of cytochrome P450 enzymes involved in monoterpene indole alkaloids biosynthesis\",\"authors\":\"Zhan Liu, Jing Pang, Yi Li, Daijing Wei, Jing Yang, Xuefei Wang, Yinggang Luo\",\"doi\":\"10.1111/ppl.14515\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Cytochrome P450 enzyme (CYP)‐catalyzed functional group transformations are pivotal in the biosynthesis of metabolic intermediates and products, as exemplified by the CYP‐catalyzed C7‐hydroxylation and the subsequent C7‐C8 bond cleavage reaction responsible for the biosynthesis of the well‐known antitumor monoterpene indole alkaloid (MIA) camptothecin. To determine the key amino acid residues responsible for the catalytic selectivity of the CYPs involved in MIA biosynthesis, we characterized the enzymes CYP72A728 and CYP72A729 as stereoselective 7‐deoxyloganic acid 7‐hydroxylases (7DLHs). We then conducted a comparative analysis of the amino acid sequences and the predicted structures of the CYP72A homologs involved in camptothecin biosynthesis, as well as those of the CYP72A homologs implicated in the pharmaceutically significant MIAs biosynthesis in <jats:italic>Catharanthus roseus</jats:italic>. The crucial amino acid residues for the catalytic selectivity of the CYP72A‐catalyzed reactions were identified through fragmental and individual residue replacement, catalytic activity assays, molecular docking, and molecular dynamic simulations analysis. The fragments 1 and 3 of CYP72A565 were crucial for its C7‐hydroxylation and C7‐C8 bond cleavage activities. Mutating fragments 1 and 2 of CYP72A565 transformed the bifunctional CYP72A565 into a monofunctional 7DLH. Evolutionary analysis of the CYP72A homologs suggested that the bifunctional CYP72A in MIA‐producing plants may have evolved into a monofunctional CYP72A. The gene pairs <jats:italic>CYP72A728‐CYP72A610</jats:italic> and <jats:italic>CYP72A729‐CYP72A565</jats:italic> may have originated from a whole genome duplication event. This study provides a molecular basis for the CYP72A‐catalyzed hydroxylation and C‐C bond cleavage activities of CYP72A565, as well as evolutionary insights of CYP72A homologs involved in MIAs biosynthesis.\",\"PeriodicalId\":20164,\"journal\":{\"name\":\"Physiologia plantarum\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":5.4000,\"publicationDate\":\"2024-09-10\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Physiologia plantarum\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1111/ppl.14515\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"PLANT SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Physiologia plantarum","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1111/ppl.14515","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"PLANT SCIENCES","Score":null,"Total":0}
Catalytic selectivity and evolution of cytochrome P450 enzymes involved in monoterpene indole alkaloids biosynthesis
Cytochrome P450 enzyme (CYP)‐catalyzed functional group transformations are pivotal in the biosynthesis of metabolic intermediates and products, as exemplified by the CYP‐catalyzed C7‐hydroxylation and the subsequent C7‐C8 bond cleavage reaction responsible for the biosynthesis of the well‐known antitumor monoterpene indole alkaloid (MIA) camptothecin. To determine the key amino acid residues responsible for the catalytic selectivity of the CYPs involved in MIA biosynthesis, we characterized the enzymes CYP72A728 and CYP72A729 as stereoselective 7‐deoxyloganic acid 7‐hydroxylases (7DLHs). We then conducted a comparative analysis of the amino acid sequences and the predicted structures of the CYP72A homologs involved in camptothecin biosynthesis, as well as those of the CYP72A homologs implicated in the pharmaceutically significant MIAs biosynthesis in Catharanthus roseus. The crucial amino acid residues for the catalytic selectivity of the CYP72A‐catalyzed reactions were identified through fragmental and individual residue replacement, catalytic activity assays, molecular docking, and molecular dynamic simulations analysis. The fragments 1 and 3 of CYP72A565 were crucial for its C7‐hydroxylation and C7‐C8 bond cleavage activities. Mutating fragments 1 and 2 of CYP72A565 transformed the bifunctional CYP72A565 into a monofunctional 7DLH. Evolutionary analysis of the CYP72A homologs suggested that the bifunctional CYP72A in MIA‐producing plants may have evolved into a monofunctional CYP72A. The gene pairs CYP72A728‐CYP72A610 and CYP72A729‐CYP72A565 may have originated from a whole genome duplication event. This study provides a molecular basis for the CYP72A‐catalyzed hydroxylation and C‐C bond cleavage activities of CYP72A565, as well as evolutionary insights of CYP72A homologs involved in MIAs biosynthesis.
期刊介绍:
Physiologia Plantarum is an international journal committed to publishing the best full-length original research papers that advance our understanding of primary mechanisms of plant development, growth and productivity as well as plant interactions with the biotic and abiotic environment. All organisational levels of experimental plant biology – from molecular and cell biology, biochemistry and biophysics to ecophysiology and global change biology – fall within the scope of the journal. The content is distributed between 5 main subject areas supervised by Subject Editors specialised in the respective domain: (1) biochemistry and metabolism, (2) ecophysiology, stress and adaptation, (3) uptake, transport and assimilation, (4) development, growth and differentiation, (5) photobiology and photosynthesis.