Identification of oxidosqualene cyclases associated with saponin biosynthesis from Astragalus membranaceus reveals a conserved motif important for catalytic function.
Identification of oxidosqualene cyclases associated with saponin biosynthesis from Astragalus membranaceus reveals a conserved motif important for catalytic function.
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DOI：
10.1016/j.jare.2022.03.014
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发表时间：
2023-01
影响因子：
10.7
通讯作者：
Qiao, Xue
中科院分区：
文献类型：
--
作者：
Chen, Kuan;Zhang, Meng;Xu, Lulu;Yi, Yang;Wang, Linlin;Wang, Haotian;Wang, Zilong;Xing, Jiangtao;Li, Pi;Zhang, Xiaohui;Shi, Xiaomeng;Ye, Min;Osbourn, Anne;Qiao, Xue
关键词：
氧化角鲨烯环化酶AmOSC 2和AmOSC 3参与了药用大豆皂苷和黄芪皂苷的生物合成。膜壳菌发现保守基序VFM/VFN是β-香树脂素/环阿屯醇脱氢酶的功能特征和产量决定因素。药用植物A.膜壳菌的特征。它们有助于黄芪中药用重要的黄芪皂苷和大豆皂苷的生物合成。保守基序VFM/VFN对于β-香树脂蛋白和环阿屯醇合酶的功能至关重要。三萜和皂苷类化合物具有广泛的药理活性。与大多数主要含有齐墩果烷型支架的豆科植物不同，黄芪不仅含有齐墩果烷型皂苷，还含有环阿屯烷型皂苷，其生物合成途径尚不清楚。本研究旨在研究氧化角鲨烯环化酶（oxidosqualene cyclases，OSCs）的功能和催化机制。膜壳菌克隆了两个OSC基因AmOSC 2和AmOSC 3。膜壳菌通过在烟草和酵母中的异源表达，以及体内瞬时表达和病毒诱导的基因沉默来研究它们的功能。利用定点突变和分子对接的方法对保守基序的催化机制进行了解释。AmOSC 2是一种β-香树脂素合成酶，在地下部分表达量较高。它与体内β-香树脂素和大豆皂苷（齐墩果烷型）的产生有关。AmOSC 3是一种在地上和地下部分都表达的环阿屯醇合酶。它与在根中合成胡萝卜苷（环阿屯烷型）和作为植物甾醇前体的环阿屯醇的合成有关。从AmOSC 2/3中，分别发现了β-香树脂素/环阿屯醇脱氢酶的保守三联体基序VFM/VFN。基序是产量的关键决定因素，如来自不同OSC的10种变体所证明的，其中含有保守基序的变体使产量增加高达12.8倍。分子对接和突变分析表明，瓦尔、Phe和Met残基共同起稳定底物的作用，其中Phe的阳离子-π相互作用起主要作用。该研究为膜荚黄芪中齐墩果烷型和环阿屯烷型三萜类化合物的生物起源提供了新的认识。保守的基序为OSC工程提供了新的机会。
Oxidosqualene cyclases AmOSC2 and AmOSC3 participated in the biosynthesis of medicinally important soyasaponins and astragalosides in A. membranaceus. Conserved motifs VFM/VFN were found as functional signatures and yield determinants for β-amyrin/cycloartenol synthases. β-Amyrin and cycloartenol synthases in the medicinal plant A. membranaceus were characterized. They contribute to the biosynthesis of medicinally important astragalosides and soyasaponins in Huang-Qi. Conserved motifs VFM/VFN are critical for the function of β-amyrin and cycloartenol synthases. Triterpenoids and saponins have a broad range of pharmacological activities. Unlike most legumes which contain mainly oleanane-type scaffold, Astragalus membranaceus contains not only oleanane-type but also cycloartane-type saponins, for which the biosynthetic pathways are unknown. This work aims to study the function and catalytic mechanism of oxidosqualene cyclases (OSCs), one of the most important enzymes in triterpenoid biosynthesis, in A. membranaceus. Two OSC genes, AmOSC2 and AmOSC3, were cloned from A. membranaceus. Their functions were studied by heterologous expression in tobacco and yeast, together with in vivo transient expression and virus-induced gene silencing. Site-directed mutagenesis and molecular docking were used to explain the catalytic mechanism for the conserved motif. AmOSC2 is a β-amyrin synthase which showed higher expression levels in underground parts. It is associated with the production of β-amyrin and soyasaponins (oleanane-type) in vivo. AmOSC3 is a cycloartenol synthase expressed in both aerial and underground parts. It is related to the synthesis of astragalosides (cycloartane-type) in the roots, and to the synthesis of cycloartenol as a plant sterol precursor. From AmOSC2/3, conserved triad motifs VFM/VFN were discovered for β-amyrin/cycloartenol synthases, respectively. The motif is a critical determinant of yield as proved by 10 variants from different OSCs, where the variant containing the conserved motif increased the yield by up to 12.8-fold. Molecular docking and mutagenesis revealed that Val, Phe and Met residues acted together to stabilize the substrate, and the cation-π interactions from Phe played the major role. The study provides insights into the biogenic origin of oleanane-type and cycloartane-type triterpenoids in Astragalus membranaceus. The conserved motif offers new opportunities for OSC engineering.
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DOI：
10.1093/bioinformatics/8.3.275
发表时间：
1992-06-01
期刊：
COMPUTER APPLICATIONS IN THE BIOSCIENCES
影响因子：
--
作者：
JONES, DT;TAYLOR, WR;THORNTON, JM
通讯作者：
THORNTON, JM
影响因子：
8.4
作者：
Reed J;Stephenson MJ;Miettinen K;Brouwer B;Leveau A;Brett P;Goss RJM;Goossens A;O'Connell MA;Osbourn A
通讯作者：
Osbourn A
影响因子：
15
作者：
Kushiro, T;Shibuya, M;Ebizuka, Y
通讯作者：
Ebizuka, Y
影响因子：
13.8
作者：
Sainsbury, Frank;Thuenemann, Eva C.;Lomonossoff, George P.
通讯作者：
Lomonossoff, George P.
黑天仙根根特异性腐胺 N-甲基转移酶的代谢特征
影响因子：
6.5
作者：
Geng, Chen;Zhao, Tengfei;Liao, Zhihua
通讯作者：
Liao, Zhihua