Department of Plant Biology, University of California, Davis, CA 95616.
Department of Chemistry, University of California, Davis, CA 95616.
Proc Natl Acad Sci U S A. 2017 Jan 31;114(5):974-979. doi: 10.1073/pnas.1612901114. Epub 2017 Jan 17.
The diversity of small molecules formed via plant diterpene metabolism offers a rich source of known and potentially new biopharmaceuticals. Among these, the microtubule-destabilizing activity of pseudolaric acid B (PAB) holds promise for new anticancer agents. PAB is found, perhaps uniquely, in the coniferous tree golden larch (Pseudolarix amabilis, Pxa). Here we describe the discovery and mechanistic analysis of golden larch terpene synthase 8 (PxaTPS8), an unusual diterpene synthase (diTPS) that catalyzes the first committed step in PAB biosynthesis. Mining of the golden larch root transcriptome revealed a large TPS family, including the monofunctional class I diTPS PxaTPS8, which converts geranylgeranyl diphosphate into a previously unknown 5,7-fused bicyclic diterpene, coined "pseudolaratriene." Combined NMR and quantum chemical analysis verified the structure of pseudolaratriene, and co-occurrence with PxaTPS8 and PAB in P amabilis tissues supports the intermediacy of pseudolaratriene in PAB metabolism. Although PxaTPS8 adopts the typical three-domain structure of diTPSs, sequence phylogeny places the enzyme with two-domain TPSs of mono- and sesqui-terpene biosynthesis. Site-directed mutagenesis of PxaTPS8 revealed several catalytic residues that, together with quantum chemical calculations, suggested a substantial divergence of PxaTPS8 from other TPSs leading to a distinct carbocation-driven reaction mechanism en route to the 5,7-trans-fused bicyclic pseudolaratriene scaffold. PxaTPS8 expression in microbial and plant hosts provided proof of concept for metabolic engineering of pseudolaratriene.
植物二萜代谢形成的小分子多样性为已知和潜在的新生物制药提供了丰富的来源。在这些小分子中，伪玉兰酸 B（PAB）的微管不稳定活性为新型抗癌药物带来了希望。PAB 存在于松科植物金松（Pseudolarix amabilis，Pxa）中，也许是独一无二的。在这里，我们描述了金松萜烯合酶 8（PxaTPS8）的发现和机制分析，这是一种不寻常的二萜合酶（diTPS），它催化 PAB 生物合成的第一步。金松根转录组的挖掘揭示了一个庞大的 TPS 家族，包括单功能类 I diTPS PxaTPS8，它将香叶基香叶基二磷酸转化为一种以前未知的 5,7-稠合双环二萜，称为“伪玉兰烯”。结合 NMR 和量子化学分析验证了伪玉兰烯的结构，并且在 P amabilis 组织中与 PxaTPS8 和 PAB 共同出现支持了伪玉兰烯在 PAB 代谢中的中间产物地位。尽管 PxaTPS8 采用了典型的 diTPS 的三结构域结构，但序列系统发育将该酶与单萜和倍半萜生物合成的二结构域 TPS 一起放置。PxaTPS8 的定点突变揭示了几个催化残基，这些残基与量子化学计算一起表明，PxaTPS8 与其他 TPS 存在很大的差异，导致在 5,7-反式稠合双环伪玉兰烯支架的形成过程中，形成了一种独特的碳正离子驱动的反应机制。在微生物和植物宿主中表达 PxaTPS8 为伪玉兰烯的代谢工程提供了概念验证。
