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Structural investigation and manipulation of key regulatory enzymes in the mevalonate pathway of isoprenoid biosynthesis

类异戊二烯生物合成甲羟戊酸途径中关键调节酶的结构研究和操作

基本信息

批准号：
10200955
负责人：
Yan Kung
金额：
$ 36.25万
依托单位：
BRYN MAWR COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10200955
关键词：
Address Affect Affinity Anabolism Architecture Binding Binding Sites Biochemical Bioinformatics Biologic Development Biological Biology C-terminal Catalysis Cholesterol Coenzyme A Complex Crystallization Development Docking Drug Targeting Engineering Enzymatic Biochemistry Enzymes Feedback High Mobility Group Proteins Human Hydroxymethylglutaryl-CoA reductase Investigation Knowledge Lead Ligand Binding Light Malaria Malignant Neoplasms Medicine Metabolic Mevalonate kinase Molecular Conformation Motion NADH NADP Natural Products Organism Pathway interactions Pharmaceutical Preparations Pharmacotherapy Post-Translational Protein Processing Production Regulation Role Site Specificity Steroids Structure Structure-Activity Relationship Testing Variant Work X-Ray Crystallography alpha helix antimicrobial drug biological systems biophysical analysis biophysical techniques cofactor comparative computer studies crosslink enzyme pathway enzyme structure human disease human pathogen in vivo inhibitor/antagonist insight isoprenoid mevalonate microbial microorganism mutant novel prevent response synthetic biology

项目摘要

PROJECT SUMMARY Isoprenoids comprise the largest and most structurally diverse class of natural products. Used as drugs in the treatment of many human diseases, isoprenoids and their derivatives are now being produced in microorganisms engineered to express the enzymes of the mevalonate pathway, which is responsible for the biosynthesis of the universal isoprenoid precursors. The key enzymes of the mevalonate pathway are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR), which catalyzes the committed and rate-limiting step, and mevalonate kinase (MK), which is a primary target for regulation of the pathway. For both of these enzymes, critical structural and mechanistic information is lacking, hindering the further development of biological systems for isoprenoid production. In particular, the structural basis of HMGR cofactor specificity for either NADH or NADPH, which differs among HMGR homologs in biology, remains unclear (Specific Aim 1). Further, a C-terminal domain of HMGR has recently been visualized in many different conformations, yet the purpose of this domain in HMGR function and catalysis is a mystery (Specific Aim 2). For MK, key features of the catalytic mechanism remain unknown due to a dearth of structural information on substrate and ligand binding (Specific Aim 3). In addition, MK is subject to feedback inhibition, where the identities and potencies of inhibitors vary widely among MK homologs, yet the structural basis governing this variance in inhibition profiles is unexplored (Specific Aim 4). In the proposed work, these gaps in fundamental structural and biochemical knowledge will be addressed using X-ray crystallography in conjunction with complementary biochemical, biophysical, and computational studies. In addition, to further probe the enzyme structure-function relationship, mutant, modified, and chimeric enzymes will be created to study the varying catalytic activities and ligand-binding features of enzymes from different organisms. Therefore, this work will not only uncover the structural determinants to key HMGR and MK functions, it will also use structure-guided protein modification to construct new enzymes with altered cofactor specificities, ligand-binding features, catalytic activities, and inhibitory responses that may be used in the biological production of isoprenoids and their derivatives. Further, by gaining deeper insight into the structural and functional differences between HMGR and MK homologs, this work may also lead to the development of new antimicrobial drugs that target mevalonate pathway enzymes in human pathogens.

项目概要类异戊二烯是最大且结构最多样化的一类天然产物。用作药物用于治疗许多人类疾病的类异戊二烯及其衍生物目前正在生产中微生物被设计来表达甲羟戊酸途径的酶，该途径负责通用类异戊二烯前体的生物合成。甲羟戊酸途径的关键酶是 3-羟基-3-甲基戊二酰辅酶 A (HMG-CoA) 还原酶 (HMGR)，催化限速步骤和甲羟戊酸激酶 (MK)，它是该途径调节的主要靶标。对于这两种酶，缺乏关键的结构和机械信息，阻碍了进一步的研究开发类异戊二烯生产的生物系统。特别是HMGR的结构基础 NADH 或 NADPH 的辅因子特异性（在生物学中 HMGR 同系物之间存在差异）仍然存在不清楚（具体目标 1）。此外，HMGR 的 C 末端结构域最近在许多不同的构象，但该结构域在 HMGR 功能和催化中的用途仍然是个谜（具体目标 2）。对于 MK，由于缺乏催化机制的关键特征仍然未知。有关底物和配体结合的结构信息（具体目标 3）。此外，MK 还受反馈抑制，MK 同系物中抑制剂的特性和效力差异很大，但控制这种抑制谱差异的结构基础尚未探索（具体目标 4）。在拟议的工作，这些基本结构和生化知识的差距将通过使用来解决 X 射线晶体学与互补的生化、生物物理和计算相结合研究。此外，为了进一步探讨酶的结构与功能关系，突变、修饰和将创建嵌合酶来研究不同的催化活性和配体结合特征来自不同生物体的酶。因此，这项工作不仅将揭示结构性决定因素 HMGR和MK的关键功能，还将利用结构引导的蛋白质修饰来构建新的酶具有改变的辅因子特异性、配体结合特征、催化活性和抑制反应可用于类异戊二烯及其衍生物的生物生产。进一步，通过深入了解深入了解 HMGR 和 MK 同系物之间的结构和功能差异，这项工作也可能导致针对人类甲羟戊酸途径酶的新型抗菌药物的开发病原体。