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.

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