Identification of oxidosqualene cyclases associated with saponin biosynthesis from Astragalus membranaceus reveals a conserved motif important for catalytic function

Chen, Kuan; Zhang, Meng; Xu, Lulu; Yang, Yi; Wang, Linlin; Wang, Haotian; Wang, Zilong; Xing, Jiangtao; Pi, Li; Zhang, Xiaohui; Shi, Xiaomeng; Ye, Min; Osbourn, Anne; Qiao, Xue

doi:10.1016/j.jare.2022.03.014

Cited by 12 publications

(17 citation statements)

References 49 publications

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“…The results of single-point mutations further indicated that F726 was the key residue for its product specificity through the cation-π interactions between Phe and intermediates (Figure 4E). 18,25,36 This is the first report on the altered product selectivity of a tricyclic triterpene synthase.…”

Section: ■ Results and Discussionmentioning

confidence: 92%

“…Previous studies have identified VFN/M motifs as key amino acids in the production of cycloartenol and β-amyrin, respectively. 25 However, the corresponding residues located at a similar position were IIH in AmAS, VFN in AtPEN1, and TFM in AtTHAS1 (Figure 2A). Because the critical residues of tricyclic triterpene synthases have rarely been studied, the residues Q256, Y258, and IIH725-727 in AmAS warrant further investigation.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…18 Recently, the triad motif VFM/N has been found to be the critical determinant for the formation of β-amyrin and cycloartenol. 25 Here we discovered the corresponding triad motif IIH in AmAS also influenced the product distribution, and Q256/Y258 played an important role in its catalytic activity. Molecular docking of AmAS or its mutant with the malabaricadienyl cation showed that Q256, Y258, and IIH725-727 are located in the active pocket and near the cation intermediate (Figure 4 and Figure S7).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Characterization of a Malabaricane-Type Triterpene Synthase from Astragalus membranaceus and Enzymatic Synthesis of Astramalabaricosides

et al. 2023

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Triterpenoids are a large and medicinally important group of natural products with a wide range of biological and pharmacological effects. Among them, malabaricane-type triterpenoids are a rare group of terpenoids with a 6,6,5-tricyclic ring system, and a few malabaricane triterpene synthases have been characterized to date. Here, an arabidiol synthase AmAS for the formation of the malabaricane-type 6,6,5-tricyclic triterpenoid skeleton in astramalabaricosides biosynthesis was characterized from Astragalus membranaceus. Multiple sequence alignment, site-directed mutagenesis, and molecular docking of AmAS reveal that residues Q256 and Y258 are essential for AmAS activity, and the triad motif IIH725-727 was the critical residue necessary for its product specificity. Mutation of IIH725-727 with VFN led to the formation of seven tricyclic, tetracyclic, and pentacyclic triterpenoids (1–7). Glycosylation of malabaricane-type triterpenoids in the biosynthesis of astramalabaricosides was also explored. Three triterpenoids (1, 5, and 6) displayed potent inhibitory effects against influenza A virus in vitro. These findings provide insights into malabaricane-type triterpenoids biosynthesis in A. membranaceus and access to diverse bioactive triterpenoids for drug discovery.

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Section: ■ Results and Discussionmentioning

confidence: 92%

Section: ■ Results and Discussionmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

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Characterization of a Malabaricane-Type Triterpene Synthase from Astragalus membranaceus and Enzymatic Synthesis of Astramalabaricosides

et al. 2023

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“…Two‐week‐old A. membranaceus seedlings were obtained as previously reported (Chen et al ., 2022) and used for gene cloning (Figure S17a). A 3‐year‐old A. membranaceus plant was collected from Daxing'anling (Heilongjiang, China) and used for chemical analysis (Figure S17b).…”

Section: Methodsmentioning

confidence: 99%

“…Though we have identified the scaffold‐forming enzyme for astragalosides (Chen et al ., 2022), the downstream steps remain unclear. Glycosylation, usually catalysed by uridine diphosphate (UDP)‐sugar‐dependent glycosyltransferases (GT), plays an important role to synthesize astragalosides (Kurze et al ., 2021; Rahimi et al ., 2019).…”

Section: Introductionmentioning

confidence: 99%

Characterization and protein engineering of glycosyltransferases for the biosynthesis of diverse hepatoprotective cycloartane‐type saponins in Astragalus membranaceus

Chen

Zhang

Gao

et al. 2023

Plant Biotechnology Journal

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Although plant secondary metabolites are important source of new drugs, obtaining these compounds is challenging due to their high structural diversity and low abundance. The roots of Astragalus membranaceus are a popular herbal medicine worldwide. It contains a series of cycloartane-type saponins (astragalosides) as hepatoprotective and antivirus components. However, astragalosides exhibit complex sugar substitution patterns which hindered their purification and bioactivity investigation. In this work, glycosyltransferases (GT) from A. membranaceus were studied to synthesize structurally diverse astragalosides. Three new GTs, AmGT1/5 and AmGT9, were characterized as 3-O-glycosyltransferase and 25-Oglycosyltransferase of cycloastragenol respectively. AmGT1 G146V/I variants were obtained as specific 3-O-xylosyltransferases by sequence alignment, molecular modelling and site-directed mutagenesis. A combinatorial synthesis system was established using AmGT1/5/9, AmGT1 G146V/S and the reported AmGT8 and AmGT8 A394F . The system allowed the synthesis of 13 astragalosides in Astragalus root with conversion rates from 22.6% to 98.7%, covering most of the sugar-substitution patterns for astragalosides. In addition, AmGT1 exhibited remarkable sugar donor promiscuity to use 10 different donors, and was used to synthesize three novel astragalosides and ginsenosides. Glycosylation remarkably improved the hepatoprotective and SARS-CoV-2 inhibition activities for triterpenoids. This is one of the first attempts to produce a series of herbal constituents via combinatorial synthesis. The results provided new biocatalytic tools for saponin biosynthesis.

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Progress on metabolites of Astragalus medicinal plants and a new factor affecting their formation: Biotransformation of endophytic fungi

Feng,

Cui

2024

Archiv der Pharmazie

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It is generally believed that the main influencing factors of plant metabolism are genetic and environmental factors. However, the transformation and catalysis of metabolic intermediates by endophytic fungi have become a new factor and resource attracting attention in recent years. There are over 2000 precious plant species in the Astragalus genus. In the past decade, at least 303 high‐value metabolites have been isolated from the Astragalus medicinal plants, including 124 saponins, 150 flavonoids, two alkaloids, six sterols, and over 20 other types of compounds. These medicinal plants contain abundant endophytic fungi with unique functions, and nearly 600 endophytic fungi with known identity have been detected, but only about 35 strains belonging to 13 genera have been isolated. Among them, at least four strains affiliated to Penicillium roseopurpureum, Alternaria eureka, Neosartorya hiratsukae, and Camarosporium laburnicola have demonstrated the ability to biotransform four saponin compounds from the Astragalus genus, resulting in the production of 66 new compounds, which have significantly enhanced our understanding of the formation of metabolites in plants of the Astragalus genus. They provide a scientific basis for improving the cultivation quality of Astragalus plants through the modification of dominant fungal endophytes or reshaping the endophytic fungal community. Additionally, they open up new avenues for the discovery of specialized, green, efficient, and sustainable biotransformation pathways for complex pharmaceutical intermediates.

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Identification of oxidosqualene cyclases associated with saponin biosynthesis from Astragalus membranaceus reveals a conserved motif important for catalytic function

Cited by 12 publications

References 49 publications

Characterization of a Malabaricane-Type Triterpene Synthase from Astragalus membranaceus and Enzymatic Synthesis of Astramalabaricosides

Characterization of a Malabaricane-Type Triterpene Synthase from Astragalus membranaceus and Enzymatic Synthesis of Astramalabaricosides

Characterization and protein engineering of glycosyltransferases for the biosynthesis of diverse hepatoprotective cycloartane‐type saponins in Astragalus membranaceus

Progress on metabolites of Astragalus medicinal plants and a new factor affecting their formation: Biotransformation of endophytic fungi

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