Malonyl-thioester Isosteres to Determine Enzyme Structure-Function Relationships - Undergrad Supplement

用丙二酰硫酯等排体确定酶的结构-功能关系 - 本科生补充材料

• 批准号: 10393793
• 负责人: Jeremy Ray Lohman
• 金额: $ 0.8万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-20 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10393793
• 关键词: Active Sites; Acyl Coenzyme A; Anabolism; Antibiotics; Antineoplastic Agents; Binding; Catalysis; Complex; Cryoelectron Microscopy; Crystallization; Drug Design; Drug Targeting; Engineering; Enzymes; Essential Fatty Acids; Fatty Acids; Fatty-acid synthase; Generations; Health; Human; Ketones; Life; Metabolic Control; Metabolic Diseases; Molecular Conformation; Process; Reaction; Research Personnel; Structure; Structure-Activity Relationship; Substrate Interaction; analog; antimicrobial; carboxylate; enzyme structure; enzyme substrate; fatty acid biosynthesis; oxetane; polyketide synthase; preservation; thioester; thioether; undergraduate student; virtual

Abstract: Malonyl-thioesters are one of the major reactive intermediates in the biosynthesis of fatty acids and polyketides. Because fatty acids are essential to cellular life, the inhibition of fatty acid synthases is a viable mechanism for the generation of antimicrobials, anticancer agents and control of metabolic disease. Polyketides on the other hand are widely used as antibiotics and anticancer agents, making polyketide synthases targets for enzyme engineering. While most intermediates in fatty acid and polyketide biosynthesis are used in reversible reactions, malonyl- thioesters are created in and used in essentially irreversible reactions. This makes studying the enzyme:malonyl-thioester interactions virtually impossible because the malonyl-thioesters are destroyed in the process. To overcome this problem analogs of malonyl-thioesters were generated by other researchers. These analogs replace the thioester ketone with a thioether or oxetane, both of which are stable to enzymatic activity. However, neither of these analogs bind in enzyme active sites in catalytically relevant orientations. Thus, there is a critical need to develop stable malonyl-thioester isosteres capable of binding in enzyme active sites to elucidate molecular interactions and conformational changes leading to efficient catalysis. The objective of this proposal is to overcome problems associated with the natural malonyl-thioesters and previously synthesized isosteres. We have a panel of malonyl-thioesters that preserve a key ketone lost in the previous isosteres. Our first aim is to solve crystal or cryo-EM structures of acyl-CoA carboxylase enzymes in complex with our best isosteres to elucidate the enzyme:substrate interactions and conformational changes. Our second aim is to solve crystal, cryo-EM or NMR structures of β-ketoacyl synthase enzymes in complex with our best isosteres to elucidate enzyme:substrate interactions and conformational changes. Together these studies will validate the use of malonyl-thioester analogs with carboxylate isosteres to capture enzyme:substrate interactions. Our structures will reveal conformational changes during catalysis that can be targeted for drug design and that need to be accounted for during enzyme engineering.

抽象的： 丙二酰thioesters是脂肪酸生物合成的主要反应性中间体之一 和聚酮化合物。因为脂肪酸对于细胞寿命至关重要，所以脂肪酸的抑制作用 合成酶是生成抗菌药物，抗癌剂和 控制代谢疾病。另一方面，聚酮化合物被广泛用作抗生素和 抗癌剂，使聚酮化合物合成酶用于酶工程。虽然大多数 脂肪酸和聚酮化合物生物合成中的中间体用于可逆反应，malonyl- 硫代植物是在本质上不可逆的反应中创建的。这使得研究 酶：Malonyl-Thioester相互作用几乎是不可能的 在此过程中被摧毁。为了克服这个问题类似物的类似物 - 由其他研究人员产生。这些类似物用硫乙醚代替硫酯酮或 氧烷，两者都稳定在酶活性上。但是，这些类似物都没有结合 在催化相关取向的酶活性位置中。那是迫切需要的 开发能够在酶活性位点结合的稳定丙二酰thioester等等法物以阐明 分子相互作用和会议变化导致有效催化。目标 该提议的内容是克服与自然丙二酰thioesters相关的问题 以前合成的等值剂。我们有一个疟疾thioesters的小组，可以保留钥匙 酮在以前的Isosteres中损失。我们的第一个目的是解决晶体或冷冻EM结构 酰基辅酶A羧化酶与我们的最佳同动作用，以阐明 酶：底物相互作用和构象变化。我们的第二个目的是解决水晶， β-酮酰基合酶的冷冻EM或NMR结构与我们的最佳等值 阐明酶：底物相互作用和构象变化。共同研究 将验证用羧酸酯等速剂使用丙二酰 - 硫酯类似物来捕获 酶：底物相互作用。我们的结构将揭示构象变化 可以针对药物设计的催化，需要在酶中考虑 工程。