Understanding Oligomer Assembly of Isoprenoid and Terpene Synthases

了解类异戊二烯和萜烯合成酶的低聚物组装

• 批准号: 10328886
• 负责人: Trey Adam Ronnebaum
• 金额: $ 1.07万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10328886
• 关键词: Adopted; Anabolism; Anti-Inflammatory Agents; Architecture; Biochemical; Biological Process; Breast Cancer Cell; Breast cancer metastasis; C-terminal; Carbon; Cells; Chemistry; Chimera organism; Clinical; Complex; Cryoelectron Microscopy; Development; Dimethylallyltranstransferase; Diphosphates; Disease; Diterpenes; Engineering; Enzymes; Exhibits; Family; Fluorescence Resonance Energy Transfer; Foundations; Geranyltranstransferase; Human; In Vitro; Incubated; Length; Link; MCF7 cell; Malignant Neoplasms; Molecular; Monitor; N-terminal; Natural Products; Nature; Penicillium; Pharmacologic Substance; Phenotype; Property; Recombinants; Research; Research Personnel; Resolution; Roentgen Rays; Role; Site-Directed Mutagenesis; Squalene Synthetase; Structure; Synthase I; Terpenes; Therapeutic; Variant; Work; analytical ultracentrifugation; anti-cancer; antimicrobial; biophysical techniques; biosynthetic product; copalyl diphosphate; dimer; ent-kaurene synthetase A; farnesyltranstransferase; fascinate; geranylgeranyl diphosphate; human disease; improved; in vivo; inhibitor; innovation; isopentenyl pyrophosphate; isoprenoid; migration; monomer; mutant; novel; novel therapeutics; polypeptide; prenylation; scaffold; starburst; terpene synthase; therapeutic target

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.

项目摘要/摘要 萜烯合酶的寡聚组件通过接近或簇改善生物合成产物通量 引导。鉴定出来自verruculosum的不寻常的二萜（C20）合酶PVCP 特征是第一个具有ABG结构结构的双功能萜烯合酶 前转移酶和II类环化酶活性。 C末端A结构域产生黄烷基凝酰二磷酸盐 然后将其在N末端BG结构域的界面上环绕至二磷酸盐。重组 表达和纯化，我确定PVCP在高浓度下以己酰胺的形式存在 - 独特 已知ABG萜烯合酶的第四纪结构 - 并以低浓度分离为单体。 有趣的是，寡聚化在丙烯酰转移酶和萜烯合酶中很常见，这些合酶具有 它们被视为单体，二聚体，三聚体，四聚体和六聚体。该提案的第一部分目的 使用PVCPS的前转移酶周期酶嵌合体确定低聚物形成的结构方面 通过使用冷冻电子显微镜（Cryo-EM）确定高分辨率结构。架构 PVCP为多功能组装线塑料生物合成提供了理想的工程机会。那， 该提案的第二部分旨在利用PVCP的低聚物组装来设计新型多功能 酶，由于许多萜烯产品暴露了有用的药物特性。这些成功完成 目标将导致开发复杂萜烯的体外生物合成多功能合酶。 最后，人前转移酶，例如Farnesyl二磷酸合酶（HFPPS），geranylgeranyly 在多种疾病中隐含了二磷酸合酶（HGGPPS）和鳞状合酶（HSQS）（HSQS） 和癌症。这些前转移酶中的每一个都被认为是体外的低聚物。但是，如果 低聚物组件是浓度依赖性的，在细胞条件下会发生低聚物组装吗？如果 那么，它的作用是什么？最后一个目标将确定低聚物组装是否发生在体内及其相关功能。