{"title":"Plant P450 forms indigo and indirubin when expressed in Escherichia coli","authors":"","doi":"10.1016/j.phytochem.2024.114268","DOIUrl":null,"url":null,"abstract":"<div><p>Indigo and indirubin are derived from indoxyl molecules, which generally occur as indoxyl glycosides in woad (<em>Isatis tinctoria</em> L.) and other indigo-producing plants. Indoxyl glycosides are biosynthesized from indole via 3-hydroxylation to form indoxyl, followed by one or more glycosylations. Enzymes that attach and remove sugars to and from indoxyl have already been isolated and characterized, while enzymes that convert indole into indoxyl in plants have remained elusive, until the identification of P450s and flavin-containing monooxygenases that hydroxylate indole. A P450 gene from woad (named CYP71B102) was heterologously expressed in <em>E. coli</em>, resulting in the formation of indigo and indirubin, as well as isatin and 2-oxindole, which along with indoxyl are putative precursors of indirubin. The addition of either isatin or 2-oxindole to the recombinant <em>E. coli</em> reduced the levels of indigo and increased the amount of indirubin, whereas coexpression of CYP71B102 with isatin hydroxylase (which degrades isatin) increased the levels of indigo and decreased the amount of indirubin, albeit slightly. The results suggest that CYP71B102 hydroxylates indole at both the 2- and 3- positions to produce 2-oxindole and indoxyl, respectively, and that the coupling of indoxyl with either 2-oxindole or isatin forms indirubin, while dimerization of indoxyl forms indigo. This P450 gene is thus likely involved in the biosynthesis of indirubin in woad, as well as the formation of indigo and its glycosidic precursors, even if other types of enzymes, such as flavin-containing monooxygenases, may be involved in indole hydroxylation in other indigo-producing plants.</p></div>","PeriodicalId":20170,"journal":{"name":"Phytochemistry","volume":null,"pages":null},"PeriodicalIF":3.2000,"publicationDate":"2024-08-30","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/S0031942224003054","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}
引用次数: 0
Abstract
Indigo and indirubin are derived from indoxyl molecules, which generally occur as indoxyl glycosides in woad (Isatis tinctoria L.) and other indigo-producing plants. Indoxyl glycosides are biosynthesized from indole via 3-hydroxylation to form indoxyl, followed by one or more glycosylations. Enzymes that attach and remove sugars to and from indoxyl have already been isolated and characterized, while enzymes that convert indole into indoxyl in plants have remained elusive, until the identification of P450s and flavin-containing monooxygenases that hydroxylate indole. A P450 gene from woad (named CYP71B102) was heterologously expressed in E. coli, resulting in the formation of indigo and indirubin, as well as isatin and 2-oxindole, which along with indoxyl are putative precursors of indirubin. The addition of either isatin or 2-oxindole to the recombinant E. coli reduced the levels of indigo and increased the amount of indirubin, whereas coexpression of CYP71B102 with isatin hydroxylase (which degrades isatin) increased the levels of indigo and decreased the amount of indirubin, albeit slightly. The results suggest that CYP71B102 hydroxylates indole at both the 2- and 3- positions to produce 2-oxindole and indoxyl, respectively, and that the coupling of indoxyl with either 2-oxindole or isatin forms indirubin, while dimerization of indoxyl forms indigo. This P450 gene is thus likely involved in the biosynthesis of indirubin in woad, as well as the formation of indigo and its glycosidic precursors, even if other types of enzymes, such as flavin-containing monooxygenases, may be involved in indole hydroxylation in other indigo-producing plants.
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.