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： 丙二酰硫酯是脂肪酸生物合成中的主要反应中间体之一 和聚酮化合物。由于脂肪酸对细胞生命至关重要，因此抑制脂肪酸 合酶是产生抗菌剂、抗癌剂和 控制代谢性疾病。另一方面，聚酮化合物广泛用作抗生素和 抗癌剂，使聚酮合酶成为酶工程的靶标。虽然大多数 脂肪酸和聚酮化合物生物合成的中间体用于可逆反应，丙二酰- 硫酯是在基本上不可逆的反应中产生和使用的。这使得学习 酶：丙二酰硫酯相互作用几乎不可能，因为丙二酰硫酯是 在此过程中被破坏。为了克服这个问题，丙二酰硫酯的类似物被开发出来。 由其他研究人员生成。这些类似物用硫醚或硫酯酮代替硫酯酮 氧杂环丁烷，两者对酶活性均稳定。然而，这些类似物都不结合 在催化相关方向的酶活性位点中。因此，迫切需要 开发能够结合酶活性位点的稳定丙二酰硫酯电子等排体以阐明 分子相互作用和构象变化导致有效的催化。目标 该提案的目的是克服与天然丙二酰硫酯相关的问题和 先前合成的等排体。我们有一组丙二酰硫酯，可以保存关键 酮在之前的等排体中丢失。我们的首要目标是解决晶体或冷冻电镜结构 酰基辅酶A羧化酶与我们最好的电子等排体复合，以阐明 酶：底物相互作用和构象变化。我们的第二个目标是解决水晶， 与我们最好的电子等排体复合的 β-酮脂酰合酶的冷冻电镜或核磁共振结构 阐明酶：底物相互作用和构象变化。这些研究一起 将验证使用丙二酰硫酯类似物与羧酸等排体来捕获 酶：底物相互作用。我们的结构将揭示构象变化 可以作为药物设计的目标并且在酶过程中需要考虑的催化作用 工程。