Malonyl-thioester Isosteres to Determine Enzyme Structure-Function Relationships

项目摘要/摘要 丙二酸硫酯是生物合成脂肪酸和聚酮的主要活性中间体之一。 由于脂肪酸对细胞生命是必不可少的，抑制脂肪酸合成酶是一种可行的机制 抗菌剂、抗癌剂的产生和代谢性疾病的控制。另一头是聚酮 手部被广泛用作抗生素和抗癌剂，使聚酮合成酶成为酶的靶标。 工程学。虽然脂肪酸和聚酮生物合成中的大多数中间体都用于可逆反应， 丙二酸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 进程。为了解决这个问题，其他研究人员合成了丙二酸硫代酯的类似物。这些 类似物用硫醚或氧杂环烷取代硫酯酮，这两种化合物对酶活性都是稳定的。 然而，这两个类似物都没有以催化相关的方向结合在酶活性部位。因此，在那里 是开发稳定的丙二酰硫酯异构体的迫切需要，该异构体能够在酶的活性部位结合 阐明导致有效催化的分子相互作用和构象变化。这样做的目的是 建议克服与天然丙二酸硫酯和先前合成的丙二酸硫酯有关的问题 等位线。我们有一组丙二酰硫代酯，它们保存了在之前的同位素组中丢失的关键酮。我们的 第一个目标是在我们最好的络合物中解决酰辅酶A羧基酶的晶体或低温EM结构 同工酶：底物相互作用和构象变化。我们的第二个目标是解决 ȕ-酮酰基合成酶与最佳异构体形成络合物的晶体、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。总之，这些研究将验证 使用丙二酸硫酯类似物和羧酸异构体捕获酶：底物相互作用。我们的 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和需要的靶点 在酶工程中需要解决的问题。

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.