Department of Biological Sciences, University of Calgary, Calgary, Canada, T2N 1N4.
Biochem J. 2012 Dec 1;448(2):261-71. doi: 10.1042/BJ20120654.
Thapsigargin is a major terpenoid constituent of Thapsia garganica root. Owing to its potent antagonistic effect on the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, thapsigargin has been widely used to study Ca2+ signalling and is also a potential drug for prostate cancer. Despite its importance, thapsigargin biosynthesis in T. garganica remains unknown. In order to decipher thapsigargin biosynthesis, deep transcript sequencing (454 and Illumina) of the T. garganica root was performed, and two terpene synthases (TgTPS1/2) were identified. Functional characterization of their encoded enzymes in a metabolically engineered yeast revealed that TgTPS1 synthesized δ-cadinene, whereas TgTPS2 produced ten distinct terpenoids. However, cultivation of the TgTPS2-expressing yeast in pH-maintained conditions (pH 6-7) yielded one major oxygenated sesquiterpenoid, suggesting that formation of multiple terpenoids was caused by acidity. The major terpene product from TgTPS2 was identified as 6β-hydroxygermacra-1(10),4-diene (kunzeaol) by mass-fragmentation pattern, retention index, the nature of its acid-induced degradation and NMR. Also, recombinant TgTPS2 efficiently catalysed the synthesis of kunzeaol in vitro from farnesyl diphosphate with a Km of 2.6 μM and a kcat of 0.03 s-1. The present paper is the first report of a kunzeaol synthase, and a mechanism for the transformation of kunzeaol into the thapsigargin backbone is proposed.
钩沙脂素是钩沙脂根的主要萜类成分。由于其对肌浆内质网 Ca2+-ATP 酶有很强的拮抗作用，钩沙脂素已被广泛用于研究 Ca2+信号转导，也是治疗前列腺癌的潜在药物。尽管它很重要，但钩沙脂素在 T. garganica 中的生物合成仍然未知。为了解析钩沙脂素的生物合成，对 T. garganica 根进行了深度转录测序（454 和 Illumina），并鉴定了两种萜烯合酶（TgTPS1/2）。在代谢工程酵母中对其编码酶的功能表征表明，TgTPS1 合成了 δ-卡烯，而 TgTPS2 则产生了十种不同的萜类化合物。然而，在 pH 维持（pH 6-7）条件下培养表达 TgTPS2 的酵母，仅产生一种主要的含氧倍半萜，这表明多种萜类化合物的形成是由酸度引起的。通过质谱-碎片模式、保留指数、酸诱导降解的性质和 NMR，从 TgTPS2 获得的主要萜烯产物被鉴定为 6β-羟基-娠二烯-1(10),4-二烯（库泽醇）。此外，重组 TgTPS2 能够从法呢基二磷酸有效地催化 kunzeaol 的体外合成，Km 值为 2.6 μM，kcat 值为 0.03 s-1。本文首次报道了一种 kunzeaol 合酶，并提出了 kunzeaol 转化为钩沙脂素骨架的机制。
