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.

摘要： 丙二酰硫酯是脂肪酸生物合成的主要反应中间体之一 和聚酮化合物。因为脂肪酸是细胞生命所必需的， 酶是产生抗微生物剂、抗癌剂和抗肿瘤剂的可行机制。 控制代谢性疾病。另一方面，聚酮化合物广泛用作抗生素， 抗癌剂，使聚酮酶成为酶工程的靶标。虽然大多数 脂肪酸和聚酮生物合成中的中间体用于可逆反应，丙二酰- 硫酯在基本上不可逆的反应中产生和使用。这使得研究 酶：丙二酰-硫酯相互作用实际上是不可能的，因为丙二酰-硫酯 在这个过程中被摧毁。为了克服这个问题， 由其他研究人员制作。这些类似物用硫醚取代硫酯酮，或 氧杂环丁烷，这两者对酶活性都是稳定的。然而，这些类似物都不结合 在酶活性位点中以催化相关的方向。因此，迫切需要 开发稳定的丙二酰硫酯电子等排体，能够结合在酶活性位点，以阐明 分子相互作用和构象变化导致有效的催化。客观 该建议的主要目的是克服与天然丙二酰基硫代酯有关的问题， 以前合成的电子等排体。我们有一个丙二酰硫酯小组， 酮在之前的电子等排体中损失。我们的第一个目标是解决晶体或cryo-EM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cryo-EM或NMR结构 阐明酶：底物相互作用和构象变化。这些研究 将验证丙二酰硫酯类似物与羧酸盐电子等排体的用途， 酶：底物相互作用。我们的结构将揭示构象变化， 催化作用，可以靶向药物设计，并需要考虑在酶的过程中， 工程.