Allylic hydroxylation of triterpenoids by a plant cytochrome P450 triggers key chemical transformations that produce a variety of bitter compounds

Takase, Shohei; Kera, Kota; Nagashima, Yoshiki; Mannen, Kazuto; Hosouchi, Tsutomu; Shinpo, Sayaka; Kawashima, Moeka; Kotake, Yuki; Yamada, Hiroki; Saga, Yusuke; Otaka, Junnosuke; Araya, Hiroshi; Kotera, Masaaki; Suzuki, Hideyuki; Kushiro, Toshio

doi:10.1074/jbc.ra119.009944

Cited by 16 publications

(5 citation statements)

References 35 publications

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“…As previously reported, HcOSC6 is the main enzyme involved in the synthesis of cucurbitacins in H. chinensis , catalyzing 2,3-oxidosqualene into cucurbitadienol, which serves as the starting substrate for CuIIa and other cucurbitacins ( Chen et al., 2022 ). Additionally, previous study found that reported 4 novel OSCs from M. charanti , related to McCBS, McIMS, McBAS and McCAS ( Takase et al., 2019 ). Moreover, three novel OSCs from T. cucumerina L. were reported in recent studies, TcBAS, TcLAS, and TcCAS, which display different activities ( Lertphadungkit et al., 2022 ).…”

Section: Discussionmentioning

confidence: 90%

“…There are a number of genes in the oxidosqualene cyclases (OSCs) family which catalyze the conversion of 2,3-oxidosqualene and its analogues, such as (3S,22S)-2,3:22,23-dioxidosqualene ( Shan et al., 2005 ; Thimmappa et al., 2014 ; Hoshino, 2017 ). The OSCs found in animals, plants, and fungi are ubiquitous ( Shibuya et al., 2007 ; Mitsuguchi et al., 2009 ; Miettinen et al., 2018 ; Takase et al., 2019 ; Lertphadungkit et al., 2022 ). Approximately 100 triterpenoid skeletons are generated by OSCs in plants, contributing to natural terpene diversity ( Xu et al., 2004 ; Suzuki et al., 2007 ; Wang et al., 2022 ).…”

Section: Introductionmentioning

confidence: 99%

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Site-directed mutagenesis identified the key active site residues of 2,3-oxidosqualene cyclase HcOSC6 responsible for cucurbitacins biosynthesis in Hemsleya chinensis

Chen

Gao

et al. 2023

Front. Plant Sci.

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Hemsleya chinensis is a Chinese traditional medicinal plant, containing cucurbitacin IIa (CuIIa) and cucurbitacin IIb (CuIIb), both of which have a wide range of pharmacological effects, including antiallergic, anti-inflammatory, and anticancer properties. However, few studies have been explored on the key enzymes that are involved in cucurbitacins biosynthesis in H. chinensis. Oxidosqualene cyclase (OSC) is a vital enzyme for cyclizing 2,3-oxidosqualene and its analogues. Here, a gene encoding the oxidosqualene cyclase of H. chinensis (HcOSC6), catalyzing to produce cucurbitadienol, was used as a template of mutagenesis. With the assistance of AlphaFold2 and molecular docking, we have proposed for the first time to our knowledge the 3D structure of HcOSC6 and its binding features to 2,3-oxidosqualene. Mutagenesis experiments on HcOSC6 generated seventeen different single-point mutants, showing that single-residue changes could affect its activity. Three key amino acid residues of HcOSC6, E246, M261 and D490, were identified as a prominent role in controlling cyclization ability. Our findings not only comprehensively characterize three key residues that are potentially useful for producing cucurbitacins, but also provide insights into the significant role they could play in metabolic engineering.

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Section: Discussionmentioning

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Site-directed mutagenesis identified the key active site residues of 2,3-oxidosqualene cyclase HcOSC6 responsible for cucurbitacins biosynthesis in Hemsleya chinensis

Chen

Gao

et al. 2023

Front. Plant Sci.

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“…Enzymes performing the C19 hydroxylation on cucurbitadienol have only been characterized in cucumber and Momordica catalyzed by the P450s CYP88L2 ( Shang et al., 2014 ) and CYP88L7 ( Takase et al., 2019 ), respectively. Despite squash being phylogenetically closer to cucumber than Momordica, there are no reports of cucurbitacins with a C19 hydroxylation in squash.…”

Section: Resultsmentioning

confidence: 99%

Metabolic engineering of cucurbitacins in Cucurbita pepo hairy roots

et al. 2022

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In this paper we show that metabolic engineering in Cucurbita pepo hairy roots can be used to both effectively increase and modify cucurbitacins. Cucurbitacins are highly-oxygenated triterpenoids originally described in the Cucurbitaceae family, but have since been found in 15 taxonomically distant plant families. Cucurbitacin B, D, E and I are the most widespread amongst the Cucurbitaceae and they have both important biological and pharmacological activities. In this study C. pepo hairy roots were used as a platform to boost production and alter the structures of the afore mentioned cucurbitacins by metabolic engineering to potentially provide new or more desirable bioactivities. We report that the ability to induce cucurbitacin biosynthesis by basic Helix-Loop-Helix transcription factors is partially conserved within the Cucurbitaceae and therefore can potentially be used as a biotechnological tool to increase cucurbitacins in several genera of this family. Additionally, overexpression of a novel acyltransferase from cucurbitacin producing Iberis amara generates a hitherto undescribed acetylation at the C3-hydroxyl group of the cucurbitadienol backbone. While overexpression of the cytochromes P450 CsCYP88L2 and McCYP88L7 from Cucumis sativus and Momordica charantia (respectively), results in accumulation of new spectral feature as revealed by High resolution liquid chromatography mass spectroscopy analysis; the m/z of the new peak supports it might be a cucurbitacin hydroxylated at the C19 position in C. pepo hairy roots. Finally, this paper is a case study of how hairy roots can be used to metabolically engineer and introduce novel modifications in metabolic pathways that have not been fully elucidated.

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“…Additionally, our tissue-speci c expression study also provides interesting information on the cucurbitacin B biosynthesis. TcCYP81B203 exhibited the highest expression in immature seeds of T. cucumerina L. and grouped into the groups of CYP81Q58, CYP81Q59, and CYP81AQ19 (Shang, et al 2014;Takase, et al 2019b) from other cucurbitaceous plants, which are related in the cucurbitacin biosynthesis. This nding supported our earlier hypothesis based on cucurbitadienol expression, which also showed the highest expression in immature seeds, although a large amount of cucurbitacin B was detected in fruit and fruit juice (Lertphadungkit, et al 2022).…”

Section: Tissue-speci C Expression and Phylogenetic Analysismentioning

confidence: 99%

A novel C-29 oxidase, CYP712D39, catalyzes the formation of bryonolic acid from isomultiflorenol in Trichosanthes cucumerina L.

Lertphadungkit,

Mekloy,

Bunsupa

2024

Preprint

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Bryonolic acid, a characteristic triterpenoid in Trichosanthes cucumerina L., is well-known due to its pharmacological activities. The biosynthetic pathway of this compound is similar to other triterpenoids, which are known to be initiated by oxidosqualene cyclase for squalene cyclization and cytochrome P450s for oxidation. However, the final step in this pathway has not been identified. This study presents a discovery of a novel enzyme, CYP712D39, with the remarkable ability to catalyze the crucial C-29 oxidation step in bryonolic acid production. We utilized previous transcriptome analysis with eight promising P450 candidates, exhibiting greater expression levels in callus tissue, which shows a high bryonolic acid production. Functional characterization experiment confirmed its capacity to convert isomultiflorenol into bryonolic acid in the WAT11 yeast system. Furthermore, we also predicted key amino acids, including F113, crucial for binding with C3-OH of isomultiflorenol for C29-oxidation. This discovery fills a critical knowledge gap and offers significant biotechnological potential for pharmaceutical and agricultural applications.

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Allylic hydroxylation of triterpenoids by a plant cytochrome P450 triggers key chemical transformations that produce a variety of bitter compounds

Cited by 16 publications

References 35 publications

Site-directed mutagenesis identified the key active site residues of 2,3-oxidosqualene cyclase HcOSC6 responsible for cucurbitacins biosynthesis in Hemsleya chinensis

Site-directed mutagenesis identified the key active site residues of 2,3-oxidosqualene cyclase HcOSC6 responsible for cucurbitacins biosynthesis in Hemsleya chinensis

Metabolic engineering of cucurbitacins in Cucurbita pepo hairy roots

A novel C-29 oxidase, CYP712D39, catalyzes the formation of bryonolic acid from isomultiflorenol in Trichosanthes cucumerina L.

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