CYP716A Subfamily Members are Multifunctional Oxidases in Triterpenoid Biosynthesis
CYP716A Subfamily Members are Multifunctional Oxidases in Triterpenoid Biosynthesis
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DOI：
10.1093/pcp/pcr146
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发表时间：
2011-12-01
影响因子：
4.9
通讯作者：
Muranaka, Toshiya
中科院分区：
文献类型：
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作者：
Fukushima, Ery O.;Seki, Hikaru;Muranaka, Toshiya
三萜类化合物是一类具有多种生物活性的次生代谢产物。齐墩果酸、熊果酸和桦木酸是植物中常见的三萜类化合物，具有抗真菌、抗菌、抗人类免疫缺陷病毒（HIV）和/或抗肿瘤等多种生物活性。在本研究中，使用蒺藜苜蓿的基因共表达分析工具，我们发现CYP 716 A12和β-香树脂素合成酶（bAS）之间存在很强的相关性，bAS编码负责2，3-氧化角鲨烯初始环化为β-香树脂素（大多数三萜皂苷的基本结构骨架）的酶。通过体外试验，我们将CYP 716 A12鉴定为β-香树脂素28-氧化酶，其能够将β-香树脂素修饰为油酸（通过赤藓二醇和可能的油酸醛）。我们还通过在内源性产生β-香树素的转基因酵母中表达CYP 716 A12证实了其体内活性。此外，还评价了CYP 716 A12的潜在α-香树脂醇和羽扇豆醇氧化活性。有趣的是，CYP 716 A12能够产生熊果酸（通过乌节醇，可能还有熊果酸）和白桦脂酸（通过白桦脂醇）。因此，CYP 716 A12被表征为具有β-香树脂醇28-氧化酶、α-香树脂醇28-氧化酶和羽扇豆醇28-氧化酶活性的多功能酶。我们还鉴定了葡萄中参与三萜类化合物生物合成的CYP 716 A12的同源物（CYP 716 A15和CYP 716 A17），这表明CYP 716 A亚家族在植物中高度保守的功能。这些研究结果将有助于异源生产植物和工业上重要的三萜类化合物，包括油酸，熊果酸和桦木酸。
Triterpenoids are a diverse group of secondary metabolites that are associated with a variety of biological activities. Oleanolic acid, ursolic acid and betulinic acid are common triterpenoids in plants with diverse biological activities, including antifungal, antibacterial, anti-human immunodeficiency virus (HIV) and/or antitumor activities. In the present study, using the gene co-expression analysis tool of Medicago truncatula, we found a strong correlation between CYP716A12 and beta-amyrin synthase (bAS), which encodes the enzyme responsible for the initial cyclization of 2,3-oxidosqualene to beta-amyrin (the basic structural backbone of most triterpenoid saponins). Through an in vitro assay, we identified CYP716A12 as a beta-amyrin 28-oxidase able to modify beta-amyrin to oleanolic acid (through erythrodiol and, possibly, oleanolic aldehyde). We also confirmed its activity in vivo, by expressing CYP716A12 in transgenic yeast that endogenously produce beta-amyrin. In addition, CYP716A12 was evaluated for its potential alpha-amyrin- and lupeol-oxidizing activities. Interestingly, CYP716A12 was able to generate ursolic acid (through uvaol and, possibly, ursolic aldehyde) and betulinic acid (through betulin). Hence, CYP716A12 was characterized as a multifunctional enzyme with beta-amyrin 28-oxidase, alpha-amyrin 28-oxidase and lupeol 28-oxidase activities. We also identified homologs of CYP716A12 in grape (CYP716A15 and CYP716A17) that are involved in triterpenoid biosynthesis, which indicates the highly conserved functionality of the CYP716A subfamily among plants. These findings will be useful in the heterologous production of pharmacologically and industrially important triterpenoids, including oleanolic acid, ursolic acid and betulinic acid.