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.

项目概要/摘要 萜烯合酶的寡聚组装通过邻近或簇提高生物合成产物通量 鉴定出来自疣状青霉的不寻常的二萜 (C20) 合酶 PvCPS。 被定性为第一个具有 abg 结构域结构的双功能萜烯合酶，其中包含 C 端 a 结构域产生香叶基香叶基二磷酸。 然后在重组后在 N 端 bg 结构域的界面处环化为二磷酸柯巴基。 表达和纯化后，我确定 PvCPS 以高浓度的六聚体形式存在 – 一种独特的 已知 abg 萜烯合酶的四级结构 - 并在低浓度下解离为单体。 阴性，寡聚化在异戊烯基转移酶和萜烯合酶中很常见，它们具有 被观察为单体、二聚体、三聚体、四聚体和六聚体。 使用 PvCPS 的异戊烯基转移酶-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。 PvCPS 为多功能装配线萜烯生物合成提供了理想的工程机会。 该提案的第二部分旨在利用 PvCPS 的低聚物组装来设计新型多功能 酶，因为许多萜产品表现出有用的药物特性。 目标将导致开发用于复杂萜烯体外生物合成的多功能合酶。 最后，人类异戊烯基转移酶，例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 然而，这些异戊烯基转移酶中的每一种都在体外被表征为寡聚物。 寡聚物组装是浓度依赖性的，寡聚物组装是否在细胞条件下发生？ 那么，它的作用是什么？最后一个目标是确定寡聚物在体内是否发生组装，以及其相关功能。