A Pair of Residues That Interactively Affect Diterpene Synthase Product Outcome.
A Pair of Residues That Interactively Affect Diterpene Synthase Product Outcome.
复制标题
DOI：
10.1021/acschembio.6b01075
复制
发表时间：
2017-03-17
影响因子：
4
通讯作者：
Peters RJ
中科院分区：
文献类型：
--
作者：
Jia M;Zhou K;Tufts S;Schulte S;Peters RJ
拉布丹相关二萜类化合物(LRD)是一个重要的天然产物超家族，其结构多样性在很大程度上取决于由I类二萜合成酶产生的碳氢骨架结构。在主要发现LRD的植物界中，相关的I类二萜合成酶显然来自所有陆地植物合成植物激素所需的Ent-Kaurene Synthase(KS)，因此通常被称为KS-like(KSL)。以前的工作是由水稻KSL的两个等位基因OsKSL5的不同功能启动的，发现了一个单一残基开关，它不仅对OsKSL5的产物产生深刻的影响，而且对更广泛的陆地植物KSS的产物也有深远的影响，具体地说，将关键的异亮氨酸替换为苏氨酸，这会中断三环阶段四环-异桂烯的形成，导致对苯二烯的产生。在这里，对这些等位基因的进一步研究导致了另一个附近的残基的发现，它调整了产品的结果。此外，新发现的残基的替代在KSS中发挥上位效应，只有在与关键异亮氨酸的替代相结合时才能改变产物分布。另一方面，这一对残基被发现对远缘蓖麻豆KSL介导的产物结果具有加性效应。因此，有可能使用这对残基的合理替代组合来显著提高(主要)选择性，以从双子叶拟南芥KS中获得新的8α-羟基-ent-pimar-15-烯产物，证明了这些结果的实用性。
The labdane-related diterpenoids (LRDs) are an important superfamily of natural products whose structural diversity critically depends on the hydrocarbon skeletal structures generated, in large part, by class I diterpene synthases. In the plant kingdom, where the LRDs are predominantly found, the relevant class I diterpene synthases are clearly derived from the ent-kaurene synthase (KS) required in all land plants for phytohormone biosynthesis and, hence, are often termed KS-like (KSL). Previous work, initiated by the distinct function of two alleles of a KSL from rice, OsKSL5, identified a single residue switch with a profound effect on not only OsKSL5 product outcome but also that of land plant KSs more broadly, specifically, replacement of a key isoleucine with threonine, which interrupts formation of the tetracyclic ent-isokaurene at the tricyclic stage, leading to production of ent-pimaradiene instead. Here, further studies of these alleles led to discovery of another, nearby residue that tunes product outcome. Substitution for this newly identified residue is additionally shown to exert an epistatic effect in KSs, altering product distribution only if combined with replacement of the key isoleucine. On the other hand, this pair of residues was found to exert additive effects on the product outcome mediated by distantly related KSLs from the eudicot castor bean. Accordingly, it was possible to use a rational combination of substitutions for this pair of residues to engineer significantly increased (dominant) selectivity for novel 8α-hydroxy-ent-pimar-15-ene product outcome in the KS from the dicot Arabidopsis thaliana, demonstrating the utility of these results.