Understanding Oligomer Assembly of Isoprenoid and Terpene Synthases

项目总结/摘要 萜烯脱氢酶的寡聚体组装通过邻近或簇合提高生物合成产物通量 通灵鉴定了来自疣状青霉的不寻常的二萜（C20）合酶PvCPS， 其特征在于是第一个具有ABG结构域结构的双功能萜烯合酶， 异戊二烯基转移酶和II类环化酶活性。C-末端a结构域生成香叶基香叶基二磷酸 其然后在N-末端bg结构域的界面处环化为柯巴基二磷酸。重组后 表达和纯化后，我确定PvCPS在高浓度下以六聚体存在-这是一种独特的 已知ABG萜烯脱氢酶的四级结构-并在低浓度下解离成单体。 有趣的是，寡聚化在异戊烯基转移酶和萜烯脱氢酶中是常见的，它们具有 作为单体、二聚体、三聚体、四聚体和六聚体观察到。本建议的第一部分旨在 使用PvCPS的异戊二烯基转移酶-环化酶嵌合体确定寡聚体形成的结构方面 通过使用冷冻电子显微镜（Cryo-EM）确定高分辨率结构。的体系结构 PvCPS为多功能装配线萜烯生物合成提供了理想的工程机会。因此，在本发明中， 该建议的第二部分旨在利用PvCPS的低聚物组装来工程化新的多功能 酶，因为许多萜烯产品表现出有用的药物性质。成功完成这些 本发明的目的将导致用于复杂萜烯体外生物合成的多功能酶的开发。 最后，人异戊烯基转移酶，如法呢基二磷酸合酶（hFPPS）、香叶基香叶基 二磷酸合酶（hGGPPS）和角鲨烯合酶（hSQS）与多种疾病有关 和癌症。这些异戊烯基转移酶中的每一种在体外都被表征为寡聚体。但如果 寡聚体组装是浓度依赖性的，寡聚体组装是否发生在细胞条件下？如果 那么它的作用是什么呢？最后一个目标将确定寡聚体组装是否在体内发生及其相关功能。

Project Summary/Abstract Oligomeric assembly of terpene synthases improves biosynthetic product flux by proximity or cluster channeling. The unusual diterpene (C20) synthase, PvCPS, from Penicillium verruculosum was identified and characterized as the first bifunctional terpene synthase with abg domain architecture containing both prenyltransferase and class II cyclase activities. The C-terminal a domain generates geranylgeranyl diphosphate which is then cyclized to copalyl diphosphate at the interface of the N-terminal bg domains. Upon recombinant expression and purification, I determined that PvCPS exists as a hexamer at high concentrations – a unique quaternary structure for known abg terpene synthases – and dissociates to monomers at low concentrations. Interestingly, oligomerization is common among prenyltransferases and terpene synthases, which have been observed as monomers, dimers, trimers, tetramers, and hexamers. The first part of this proposal aims to determine the structural aspects of oligomer formation using the prenyltransferase-cyclase chimera of PvCPS by determining the high-resolution structure using Cryo-Electron Microscopy (Cryo-EM). The architecture of PvCPS presents an ideal engineering opportunity for multifunctional assembly-line terpene biosynthesis. Thus, the second part of this proposal aims to utilize the oligomer assembly of PvCPS to engineer novel multifunctional enzymes, since many terpene products exhibit useful pharmaceutical properties. Successful completion of these aims will lead to the development of multifunctional synthases for in vitro biosynthesis of complex terpenes. Lastly, human prenyltransferases, such as farnesyl diphosphate synthase (hFPPS), geranylgeranyl diphosphate synthase (hGGPPS), and squalene synthase (hSQS), have been implicated in a variety of diseases and cancers. Each of these prenyltransferases has been characterized as an oligomer in vitro. However, if oligomer assembly is concentration-dependent, does oligomer assembly occur under cellular conditions? And if so, what is its role? The last aim will determine if oligomer assembly occurs in vivo, and its associated function.