Structure and Function of Biosynthetic Enzymes

生物合成酶的结构和功能

基本信息

批准号：
10700146
负责人：
DAVID W CHRISTIANSON
金额：
$ 35.15万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-08-01 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10700146
关键词：
Active Sites Alcohols Anabolism Anti-Bacterial Agents Anti-Inflammatory Agents Antifungal Agents Antimalarials Antineoplastic Agents Antioxidants Artemisinins Aspergillus flavus Binding Biology Canes Carbon Catalysis Catalytic Domain Chemicals Chemistry Chemotherapy-Oncologic Procedure Complex Cryoelectron Microscopy Cyclization Data Dimethylallyltranstransferase Engineering Enzymatic Biochemistry Enzymes Evolution Exhibits Family Foundations Funding Gibberellins Hydroxyl Radical Investigation Kinetics Lead Length Life Link Malignant Neoplasms Medicine Molecular Mutagenesis Natural Products Natural Products Chemistry Nature Paclitaxel Pathway interactions Penicillium Periodicity Pharmaceutical Preparations Physical condensation Planet Earth Property Protein Engineering Proteins Protons Reaction Reporting Research Roentgen Rays Services Sesquiterpenes Structure Structure-Activity Relationship Terpenes Terpenoid Biosynthesis Pathway Therapeutic Time Water analog bisphosphonate catalyst central nervous system injury chemical reaction drug discovery enhancing factor ent-kaurene synthetase A fusicoccin inhibitor manufacture novel peptide synthase pharmacologic reaction rate scaffold structural biology synthetic biology terpene synthase

项目摘要

The proposed research explores the structure and mechanism of terpene cyclases, which are unique among enzymes in that they catalyze the most complex carbon-carbon bond forming reactions in nature: on average, more than half of the substrate carbon atoms undergo changes in bonding and/or hybridization during the course of a typical enzyme-catalyzed reaction. Notably, many terpenoids exhibit useful pharmacological properties, such as the blockbuster cancer chemotherapy drug Taxol (paclitaxel) and the antimalarial drug artemisinin. Thus, a better understanding of terpene cyclase structure and mechanism will enable drug discovery and manufacturing at the interface of natural products chemistry, enzymology, structural biology, and synthetic biology. To advance our understanding of structure-function relationships in terpene cyclases, we will pursue the following lines of investigation: (1) We will determine the structural basis of substrate binding, transit, and catalysis in a class I assembly- line terpene synthase, fusicoccadiene synthase from Phomopsis amygdali (PaFS). We will determine cryo-EM structures of PaFS complexes with an inhibitor and with a substrate analogue, and we will determine the influence of oligomeric structure as well as the interdomain linker on substrate channeling between the prenyltransferase and cyclase domains. These studies will broaden our understanding of substrate channeling – perhaps better designated as "directed substrate transit" – between covalently-linked enzymes catalyzing consecutive reactions in a biosynthetic pathway. (2) We will determine the structural basis of substrate binding, transit, and catalysis in class II assembly- line terpene synthases, the copalyl diphosphate synthases from Penicillium verruculosum (PvCPS) and Penicillium fellutanum (PfCPS). We will complete the cryo-EM structure determination of PfCPS, and we will determine the cryo-EM structures of its complexes with a substrate analogue and product. We will also determine whether directed substrate transit occurs between the prenyltransferase and cyclase domains in both PfCPS and PvCPS, and we will ascertain the importance of oligomeric structure for catalytic function. (3) We will explore and exploit the structural basis of chemodiversity in terpene biosynthesis, focusing on sesquiterpene synthases that quench reactive carbocation intermediates with hydroxyl or amino nucleophiles. We will convert our paradigm for protein engineering, epi-isozizaene synthase, into a sesquiterpene alcohol synthase. We will also determine structure-function relationships for the sesquiterpene synthase FlvF from Aspergillus flavus to understand how it catalyzes the condensation of a cyclic sesquiterpene with dimethylcadaverine. Importantly, FlvF represents the first example of a synthase that catalyzes C–N bond formation with a cyclic terpene.

拟议的研究探讨了萜烯循环的结构和机制，这是独特的在酶中，它们催化自然界中最复杂的碳碳键形成反应：平均，超过一半的底物碳原子在粘合和/或杂交期间经历了变化典型的酶催化反应的过程。值得注意的是，许多萜类化合物暴露了有用的药物。特性，例如重磅癌化学疗法药物紫杉醇（紫杉醇）和抗疟药阿耳马素。这是对萜烯环化酶结构和机制的更好理解将使药物在天然产品化学，酶学，结构生物学和合成生物学。为了促进我们对萜烯循环中结构功能关系关系的理解，我们将追求以下调查措施：（1）我们将确定I类组装中底物结合，过境和催化的结构基础 - 萜烯合酶，来自Phomopsis杏仁核（PAFS）的fusicocadiene合酶。我们将确定低温EM PAFS复合物与抑制剂和底物类似物的结构，我们将确定寡聚结构以及域间接头对底物引导的影响前转移酶和环化酶结构域。这些研究将扩大我们对底物渠道的理解 - 也许更好地设计为“定向底物过境” - 共价连接的酶催化生物合成途径中的连续反应。（2）我们将确定II类组装中底物结合，过境和催化的结构基础 - 线萜烯合酶，来自verruculosum（PVCPS）和青霉（PFCPS）。我们将完成PFCP的冷冻EM结构确定，我们将用底物类似物和产物确定其复合物的冷冻EM结构。我们也会确定是否在前转移酶和环域域之间发生定向底物转运 PFCP和PVCP都可以确定寡聚结构对催化功能的重要性。（3）我们将探索和探索萜烯生物合成中化学多样性的结构基础，重点是溶解反应性碳定位中间的倍半萜合酶与羟基或氨基核硫杆菌。我们将转化用于蛋白质工程的范式Epi-Isozizaene合酶，为倍半萜醇合酶。我们还将确定倍半萜合酶FLVF的结构功能关系曲霉曲霉，以了解其如何催化循环倍半萜与与 Dimethylcadaverine。重要的是，FLVF代表了催化C – N键的合酶的第一个例子用环状萜烯形成。

项目成果

期刊论文数量（47）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Biochemistry. Five golden rings.

生物化学。

DOI：
10.1126/science.1125298
发表时间：
2006
期刊：
Science (New York, N.Y.)
影响因子：
0
作者：
Christianson,DavidW
通讯作者：
Christianson,DavidW

Probing the Role of Active Site Water in the Sesquiterpene Cyclization Reaction Catalyzed by Aristolochene Synthase.

DOI：
10.1021/acs.biochem.6b00343
发表时间：
2016-05-24
期刊：
Biochemistry
影响因子：
2.9
作者：
Chen M;Chou WK;Al-Lami N;Faraldos JA;Allemann RK;Cane DE;Christianson DW
通讯作者：
Christianson DW

Exploring the Influence of Domain Architecture on the Catalytic Function of Diterpene Synthases.

DOI：
10.1021/acs.biochem.7b00137
发表时间：
2017-04-11
期刊：
Biochemistry
影响因子：
2.9
作者：
Pemberton TA;Chen M;Harris GG;Chou WK;Duan L;Köksal M;Genshaft AS;Cane DE;Christianson DW
通讯作者：
Christianson DW

Probing the mechanism of 1,4-conjugate elimination reactions catalyzed by terpene synthases.

DOI：
10.1021/ja311022s
发表时间：
2012-12-26
期刊：
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
影响因子：
15
作者：
Faraldos, Juan A.;Gonzalez, Veronica;Li, Amang;Yu, Fanglei;Koeksal, Mustafa;Christianson, David W.;Allemann, Rudolf K.
通讯作者：
Allemann, Rudolf K.

Role of arginine-304 in the diphosphate-triggered active site closure mechanism of trichodiene synthase.