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： 丙二酸硫酯是生物合成脂肪酸的主要活性中间体之一。 和聚酮类化合物。因为脂肪酸对细胞生命是必不可少的，所以脂肪酸的抑制 合成酶是一种可行的机制，用于产生抗微生物、抗癌药物和 控制代谢性疾病。另一方面，聚酮类化合物被广泛用作抗生素和 抗癌剂，使聚酮合成酶成为酶工程的靶标。虽然大多数人 脂肪酸和聚酮生物合成的中间体用于可逆反应，丙二酸基- 硫代酯是在基本不可逆的反应中生成和使用的。这使得学习成为 酶：丙二酰硫酯相互作用几乎是不可能的，因为丙二酰硫酯 在这个过程中被摧毁了。为了解决这个问题，丙二酸硫代酯的类似物是 由其他研究人员产生。这些类似物用硫醚或硫醚取代硫酯酮 氧杂环己烷，两者对酶活性都是稳定的。然而，这两个类比都不会绑定 在催化相关取向的酶活性部位。因此，迫切需要 开发能够与酶活性部位结合的稳定的丙二酰硫酯异构体来阐明 分子相互作用和构象变化导致有效的催化作用。目标是 这项建议的目的是克服与天然丙二酸硫酯和 先前合成的同种异位体。我们有一组丙二酰硫代酯可以保存一把钥匙 在之前的同位素线中丢失的酮。我们的第一个目标是解决晶体或低温电磁结构 酰辅酶A羧基酶与我们最好的同工酶的复合体 酶：底物相互作用和构象变化。我们的第二个目标是解开水晶， β-酮酰基合成酶与我们最好的同位素络合物的低温电子显微镜或核磁共振结构 阐明酶：底物相互作用和构象变化。把这些研究放在一起 将验证丙二酸硫酯类似物与羧酸异构体的使用以捕获 酶：底物相互作用。我们的结构将显示构象变化 可作为药物设计靶点的催化作用，需要在酶促反应过程中考虑 工程学。