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

 描述（由申请人提供）：类异戊二烯代表最大和结构最多样化的天然产物类别。用作治疗多种人类疾病的药物，类异戊二烯及其衍生物现在可以在表达甲羟戊酸途径的酶的微生物中产生，所述酶负责类异戊二烯前体的生物合成。体内甲羟戊酸途径调节的酶靶是3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶（HMGR）和甲羟戊酸激酶（MK）。对于这两种酶，目前缺乏关键的结构和机制信息，阻碍了微生物系统的进一步发展，类异戊二烯生产。具体地说，HMGR辅因子对NADH或NADPH的特异性的结构基础在HMGR同源物中是不同的，目前还不清楚。对于受到紧密反馈抑制的MK，抑制剂的身份及其抑制效力在来自不同生物体的MK中变化很大，但控制抑制谱中这种变化的结构基础完全未被探索。在这项研究中，这些基本结构知识的差距将直接解决。使用X射线晶体学结合互补的生化研究，HMGR辅因子特异性和MK抑制酶同系物之间的差异的结构基础进行了研究。此外，为了进一步探索酶的结构-功能关系，将构建和研究嵌合酶，以改变或切换来自不同生物体的酶之间的差异活性。因此，拟议的工作不仅将揭示关键HMGR和MK特性的结构决定因素，这些特性尚未得到研究，还将采用结构指导的蛋白质修饰来产生具有更理想的辅因子偏好和抑制反应的新酶，这些酶可用于类异戊二烯天然产物的微生物生产。

 DESCRIPTION (provided by applicant): Isoprenoids represent the largest and most structurally diverse class of natural products. Used as drugs for the treatment of a multitude of human diseases, isoprenoids and their derivatives can now be produced in microorganisms expressing the enzymes of the mevalonate pathway, which is responsible for the biosynthesis of isoprenoid precursors. The enzyme targets of in vivo mevalonate pathway regulation are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR) and mevalonate kinase (MK). For both of these enzymes, key structural and mechanistic information is currently lacking, hindering the further development of microbial systems for isoprenoid production. Specifically, the structural basis of HMGR cofactor specificity for either NADH or NADPH, which differs among HMGR homologs, is as yet unknown. For MK, which is subject to tight feedback inhibition, the identity of the inhibitors and their inhibition potencies vary widely across MKs frm different organisms, yet the structural underpinnings that govern such a variance in inhibition profiles are entirely unexplored. In this study, these gaps in fundamental structural knowledge will be addressed directly. Using X-ray crystallography in conjunction with complementary biochemical studies, the structural bases of both HMGR cofactor specificity and MK inhibition variance among enzyme homologs will be investigated. In addition, to further probe the enzyme structure-function relationship, chimeric enzymes will be constructed and studied in order to alter or switch the differential activities between enzymes from different organisms. Therefore, the proposed work will not only reveal the structural determinants to critical HMGR and MK properties that remain unstudied, it will also employ structure-guided protein modification to yield new enzymes with more desirable cofactor preferences and inhibitory responses that can be used in the microbial production of isoprenoid natural products.