Malonyl-thioester Isosteres to Determine Enzyme Structure-Function Relationships

丙二酰硫酯电子等排体测定酶的结构-功能关系

基本信息

批准号：
10453633
负责人：
Jeremy Ray Lohman
金额：
$ 30.35万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-20 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10453633
关键词：
Acetyl-CoA Carboxylase Active Sites Acyl Carrier Protein Acyl Coenzyme A Address Allosteric Regulation Anabolism Antibiotics Antineoplastic Agents Binding Biological Biotin Biotin carboxylase Carrier Proteins Catalysis Complex Cryoelectron Microscopy Crystallization Crystallography Development Drug Design Drug Targeting Electrostatics Engineering Enzymes Essential Drugs Essential Fatty Acids Fatty Acids Fatty-acid synthase Futile Cycling Generations Goals Health Human Hydrogen Bonding Hydrophobic Interactions Ketones Knowledge Lead Learning Life Malonyl Coenzyme A Metabolic Control Metabolic Diseases Molecular Molecular Conformation Outcome Pattern Pharmaceutical Preparations Positioning Attribute Process Reaction Research Research Personnel Route Structure Structure-Activity Relationship Substrate Interaction Substrate Specificity System Techniques analog antimicrobial biophysical analysis carboxylate combinatorial drug action enzyme structure enzyme substrate fatty acid biosynthesis improved novel oxetane polyketide synthase preservation structural biology thioester thioether virtual

项目摘要

Project Summary/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 ȕ-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.

项目概要/摘要丙二酰硫酯是脂肪酸和聚酮化合物生物合成中的主要反应中间体之一。由于脂肪酸对细胞生命至关重要，因此抑制脂肪酸合酶是一种可行的机制。另一方面，抗菌药物、抗癌药物的产生和代谢疾病的控制。手被广泛用作抗生素和抗癌剂，使聚酮合酶成为酶的靶标虽然脂肪酸和聚酮化合物生物合成中的大多数中间体用于可逆反应，丙二酰硫酯是在本质上不可逆的反应中产生和使用的，这使得研究成为可能。酶：丙二酰硫酯相互作用实际上是不可能的，因为丙二酰硫酯在酶中被破坏为了克服这个问题，其他研究人员产生了丙二酰硫酯的类似物。类似物用硫醚或氧杂环丁烷代替硫酯酮，这两种物质对酶活性都稳定。然而，这些类似物都不以催化相关方向结合在酶活性位点上。迫切需要开发能够在酶活性位点结合的稳定的丙二酰硫酯电子等排体阐明导致有效催化的分子相互作用和构象变化。该提案旨在克服与天然丙二酰硫酯和先前合成的相关问题我们有一组丙二酰硫酯，可以保留我们之前的等排体中丢失的关键酮。第一个目标是用我们最好的技术解决酰基辅酶A羧化酶复合物的晶体或冷冻电镜结构阐明酶的等排体：底物相互作用和构象变化我们的第二个目标是解决。与我们最好的电子等排体复合的 β-酮脂酰合酶的晶体、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，这些研究将共同验证。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。结构将揭示催化过程中的构象变化，这些变化可以作为药物设计的目标，并且需要在酶工程过程中要考虑在内。