Identification and Functional Characterization of Monofunctional ent-Copalyl Diphosphate and ent-Kaurene Synthases in White Spruce Reveal Different Patterns for Diterpene Synthase Evolution for Primary and Secondary Metabolism in Gymnosperms
Identification and Functional Characterization of Monofunctional ent-Copalyl Diphosphate and ent-Kaurene Synthases in White Spruce Reveal Different Patterns for Diterpene Synthase Evolution for Primary and Secondary Metabolism in Gymnosperms
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DOI：
10.1104/pp.109.151456
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发表时间：
2010-03-01
期刊：
影响因子：
7.4
通讯作者：
Bohlmann, Joerg
中科院分区：
文献类型：
--
作者：
Keeling, Christopher I.;Dullat, Harpreet K.;Bohlmann, Joerg
四环二萜对映贝壳杉烯的生物合成是赤霉素激素一般（初级）代谢的关键步骤。对映-贝壳杉烯是通过中间体对映-柯巴基二磷酸香叶基香叶基二磷酸的两步环化形成的。在低地植物中，苔藓小立碗藓，一个单一的双功能二萜合酶（diTPS）催化这两个步骤。相反，在被子植物中，两个连续的环化由两个不同的单功能酶催化，对映-柯巴基二磷酸合酶（CPS）和对映-贝壳杉烯合酶（KS）。裸子植物中负责内贝壳杉烯生物合成的一种或多种酶一直难以捉摸。然而，一些专门的（次级）代谢的双功能diTPS先前已经在裸子植物中被表征，并且所有已知的用于针叶树中树脂酸生物合成的diTPS都是双功能的。为了进一步了解对映贝壳杉烯生物合成的演变以及裸子植物中一般和专门的二萜类代谢的演变，我们着手确定针叶树是否使用单一的双功能diTPS或两个单功能diTPS在对映贝壳杉烯途径。利用表达序列标签、全长cDNA、基因组DNA和靶向细菌人工染色体测序的组合，我们鉴定了来自白色云杉（Picea glauca）的两个候选CPS和KS基因及其在锡特卡云杉（Picea sitchensis）中的直系同源物。重组酶的功能表征表明，白色云杉中对映贝壳杉烯的生物合成是由两种单功能的双TPS PgCPS和PgKS催化的。基因结构和酶功能的比较分析突出了这些diTPSs的分子进化在裸子植物和被子植物之间是保守的。相反，在被子植物和裸子植物中，用于专门代谢的diTPS进化不同。
The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene synthase (diTPS) catalyzes both steps. In contrast, in angiosperms, the two consecutive cyclizations are catalyzed by two distinct monofunctional enzymes, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). The enzyme, or enzymes, responsible for ent-kaurene biosynthesis in gymnosperms has been elusive. However, several bifunctional diTPS of specialized (secondary) metabolism have previously been characterized in gymnosperms, and all known diTPSs for resin acid biosynthesis in conifers are bifunctional. To further understand the evolution of ent-kaurene biosynthesis as well as the evolution of general and specialized diterpenoid metabolisms in gymnosperms, we set out to determine whether conifers use a single bifunctional diTPS or two monofunctional diTPSs in the ent-kaurene pathway. Using a combination of expressed sequence tag, full-length cDNA, genomic DNA, and targeted bacterial artificial chromosome sequencing, we identified two candidate CPS and KS genes from white spruce (Picea glauca) and their orthologs in Sitka spruce (Picea sitchensis). Functional characterization of the recombinant enzymes established that ent-kaurene biosynthesis in white spruce is catalyzed by two monofunctional diTPSs, PgCPS and PgKS. Comparative analysis of gene structures and enzyme functions highlights the molecular evolution of these diTPSs as conserved between gymnosperms and angiosperms. In contrast, diTPSs for specialized metabolism have evolved differently in angiosperms and gymnosperms.