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

项目摘要 类异戊二烯包括最大和结构最多样化的天然产物类别。作为毒品， 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 微生物工程化以表达甲羟戊酸途径的酶，其负责 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），其催化承诺的 和限速步骤，以及甲羟戊酸激酶（MK），其是调节该途径的主要靶标。 对于这两种酶，缺乏关键的结构和机制信息，阻碍了进一步的研究。 开发用于类异戊二烯生产的生物系统。特别是，HMGR的结构基础 辅酶对NADH或NADPH的特异性，在生物学上与HMGR同源物不同， 不明确（具体目标1）。此外，HMGR的C-末端结构域最近已经在许多研究中被可视化。 不同的构象，但这个域在HMGR功能和催化中的目的是一个谜 （具体目标2）。对于MK，催化机制的关键特征仍然未知，因为缺乏 底物和配体结合的结构信息（具体目标3）。此外，MK还受到 反馈抑制，其中抑制剂的特性和效力在MK同系物之间变化很大，然而 控制抑制谱中这种差异的结构基础尚未探索（具体目标4）。在 建议的工作，这些差距的基本结构和生物化学知识将得到解决，使用 X射线晶体学与互补的生物化学、生物物理学和计算 问题研究此外，为了进一步探索酶的结构与功能的关系，对突变体、修饰体和 嵌合酶将被创建，以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， 关键HMGR和MK功能，它还将使用结构指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。此外，通过深入 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 导致靶向人体内甲羟戊酸途径酶的新的抗微生物药物的开发 病原体

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.