Wen-Ke Xu , Chen-Xiao Zhao , Xiao-Wen Yang , Yue-Gui Chen , Li-Ping Long , Yuan-Feng Yan , Kai Guo , Sheng-Hong Li , Yan Liu
{"title":"应用于稀有麦冬苷和人参皂苷生物合成的巴黎多花植物糖基转移酶的特征。","authors":"Wen-Ke Xu , Chen-Xiao Zhao , Xiao-Wen Yang , Yue-Gui Chen , Li-Ping Long , Yuan-Feng Yan , Kai Guo , Sheng-Hong Li , Yan Liu","doi":"10.1016/j.phytochem.2024.114173","DOIUrl":null,"url":null,"abstract":"<div><p>Saponins are bioactive components of many medicinal plants, possessing complicated chemical structures and extensive pharmacological activities, but the production of high-value saponins remains challenging. In this study, a 6′-<em>O</em>-glucosyltransferase <em>Pp</em>UGT7 (<em>Pp</em>UGT91AH7) was functionally characterized from <em>Paris polyphylla</em> Smith var. <em>y</em><em>unnanensis</em> (Franch.) Hand. -Mazz., which can transfer a glucosyl group to the C-6′ position of diosgenin-3-<em>O</em>-rhamnosyl-(1 → 2)-glucoside (<strong>1</strong>), pennogenin-3-<em>O</em>-rhamnosyl-(1 → 2)-glucoside (<strong>2</strong>), and diosgenin-3-<em>O</em>-glucoside (<strong>5</strong>). The <em>K</em><sub>M</sub> and <em>K</em><sub>cat</sub> values of <em>Pp</em>UGT7 towards the substrate <strong>2</strong> were 8.4 μM and 2 × 10<sup>−3</sup> s<sup>−1</sup>, respectively. Through molecular docking and site-directed mutagenesis, eight residues were identified to interact with the sugar acceptor <strong>2</strong> and be crucial for enzyme activity. Moreover, four rare ophiopogonins and ginsenosides were obtained by combinatorial biosynthesis, including an undescribed compound ruscogenin-3-<em>O</em>-glucosyl-(1 → 6)-glucoside (<strong>10</strong>). Firstly, two monoglycosides <strong>9</strong> and <strong>11</strong> were generated using a known sterol 3-<em>O</em>-<em>β</em>-glucosyltransferase <em>Pp</em>UGT80A40 with ruscogenin (<strong>7</strong>) and 20(<em>S</em>)-protopanaxadiol (<strong>8</strong>) as substrates, which were further glycosylated to the corresponding diglycosides <strong>10</strong> and <strong>12</strong> under the catalysis of <em>Pp</em>UGT7. In addition, compounds <strong>7</strong>–<strong>11</strong> were found to show inhibitory effects on the secretion of TNF-<strong><em>α</em></strong> and IL-6 in macrophages RAW264.7. The findings provide valuable insights into the enzymatic glycosylation processes in the biosynthesis of bioactive saponins in <em>P. polyphylla</em> var. <em>yunnanensis</em>, and also serve as a reference for utilizing UDP-glycosyltransferases to construct high-value or rare saponins for development of new therapeutic agents.</p></div>","PeriodicalId":20170,"journal":{"name":"Phytochemistry","volume":null,"pages":null},"PeriodicalIF":3.2000,"publicationDate":"2024-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Characterization of a glycosyltransferase from Paris polyphylla for application in biosynthesis of rare ophiopogonins and ginsenosides\",\"authors\":\"Wen-Ke Xu , Chen-Xiao Zhao , Xiao-Wen Yang , Yue-Gui Chen , Li-Ping Long , Yuan-Feng Yan , Kai Guo , Sheng-Hong Li , Yan Liu\",\"doi\":\"10.1016/j.phytochem.2024.114173\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Saponins are bioactive components of many medicinal plants, possessing complicated chemical structures and extensive pharmacological activities, but the production of high-value saponins remains challenging. In this study, a 6′-<em>O</em>-glucosyltransferase <em>Pp</em>UGT7 (<em>Pp</em>UGT91AH7) was functionally characterized from <em>Paris polyphylla</em> Smith var. <em>y</em><em>unnanensis</em> (Franch.) Hand. -Mazz., which can transfer a glucosyl group to the C-6′ position of diosgenin-3-<em>O</em>-rhamnosyl-(1 → 2)-glucoside (<strong>1</strong>), pennogenin-3-<em>O</em>-rhamnosyl-(1 → 2)-glucoside (<strong>2</strong>), and diosgenin-3-<em>O</em>-glucoside (<strong>5</strong>). The <em>K</em><sub>M</sub> and <em>K</em><sub>cat</sub> values of <em>Pp</em>UGT7 towards the substrate <strong>2</strong> were 8.4 μM and 2 × 10<sup>−3</sup> s<sup>−1</sup>, respectively. Through molecular docking and site-directed mutagenesis, eight residues were identified to interact with the sugar acceptor <strong>2</strong> and be crucial for enzyme activity. Moreover, four rare ophiopogonins and ginsenosides were obtained by combinatorial biosynthesis, including an undescribed compound ruscogenin-3-<em>O</em>-glucosyl-(1 → 6)-glucoside (<strong>10</strong>). Firstly, two monoglycosides <strong>9</strong> and <strong>11</strong> were generated using a known sterol 3-<em>O</em>-<em>β</em>-glucosyltransferase <em>Pp</em>UGT80A40 with ruscogenin (<strong>7</strong>) and 20(<em>S</em>)-protopanaxadiol (<strong>8</strong>) as substrates, which were further glycosylated to the corresponding diglycosides <strong>10</strong> and <strong>12</strong> under the catalysis of <em>Pp</em>UGT7. In addition, compounds <strong>7</strong>–<strong>11</strong> were found to show inhibitory effects on the secretion of TNF-<strong><em>α</em></strong> and IL-6 in macrophages RAW264.7. The findings provide valuable insights into the enzymatic glycosylation processes in the biosynthesis of bioactive saponins in <em>P. polyphylla</em> var. <em>yunnanensis</em>, and also serve as a reference for utilizing UDP-glycosyltransferases to construct high-value or rare saponins for development of new therapeutic agents.</p></div>\",\"PeriodicalId\":20170,\"journal\":{\"name\":\"Phytochemistry\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.2000,\"publicationDate\":\"2024-06-06\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Phytochemistry\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0031942224002103\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Phytochemistry","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0031942224002103","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Characterization of a glycosyltransferase from Paris polyphylla for application in biosynthesis of rare ophiopogonins and ginsenosides
Saponins are bioactive components of many medicinal plants, possessing complicated chemical structures and extensive pharmacological activities, but the production of high-value saponins remains challenging. In this study, a 6′-O-glucosyltransferase PpUGT7 (PpUGT91AH7) was functionally characterized from Paris polyphylla Smith var. yunnanensis (Franch.) Hand. -Mazz., which can transfer a glucosyl group to the C-6′ position of diosgenin-3-O-rhamnosyl-(1 → 2)-glucoside (1), pennogenin-3-O-rhamnosyl-(1 → 2)-glucoside (2), and diosgenin-3-O-glucoside (5). The KM and Kcat values of PpUGT7 towards the substrate 2 were 8.4 μM and 2 × 10−3 s−1, respectively. Through molecular docking and site-directed mutagenesis, eight residues were identified to interact with the sugar acceptor 2 and be crucial for enzyme activity. Moreover, four rare ophiopogonins and ginsenosides were obtained by combinatorial biosynthesis, including an undescribed compound ruscogenin-3-O-glucosyl-(1 → 6)-glucoside (10). Firstly, two monoglycosides 9 and 11 were generated using a known sterol 3-O-β-glucosyltransferase PpUGT80A40 with ruscogenin (7) and 20(S)-protopanaxadiol (8) as substrates, which were further glycosylated to the corresponding diglycosides 10 and 12 under the catalysis of PpUGT7. In addition, compounds 7–11 were found to show inhibitory effects on the secretion of TNF-α and IL-6 in macrophages RAW264.7. The findings provide valuable insights into the enzymatic glycosylation processes in the biosynthesis of bioactive saponins in P. polyphylla var. yunnanensis, and also serve as a reference for utilizing UDP-glycosyltransferases to construct high-value or rare saponins for development of new therapeutic agents.
期刊介绍:
Phytochemistry is a leading international journal publishing studies of plant chemistry, biochemistry, molecular biology and genetics, structure and bioactivities of phytochemicals, including ''-omics'' and bioinformatics/computational biology approaches. Phytochemistry is a primary source for papers dealing with phytochemicals, especially reports concerning their biosynthesis, regulation, and biological properties both in planta and as bioactive principles. Articles are published online as soon as possible as Articles-in-Press and in 12 volumes per year. Occasional topic-focussed special issues are published composed of papers from invited authors.