Malonyl-thioester Isosteres to Determine Enzyme Structure-Function Relationships

项目总结/摘要 丙二酰硫酯是脂肪酸和聚酮生物合成的主要反应中间体之一。 因为脂肪酸是细胞生命所必需的，所以脂肪酸脱氢酶的抑制是细胞死亡的可行机制。 产生抗菌剂、抗癌剂和控制代谢疾病。另一方面， 手被广泛用作抗生素和抗癌剂，使聚酮酶成为酶的靶标 工程.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， 丙二酸硫酯是在基本不可逆的反应中产生和使用的。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-硫酯在酶中被破坏。 过程为了克服这个问题，其他研究人员产生了丙二酰硫酯的类似物。这些 类似物用硫醚或氧杂环丁烷代替硫酯酮，硫醚或氧杂环丁烷两者都对酶活性稳定。 然而，这两种类似物都不能以催化相关的方向结合在酶活性位点上。因此 迫切需要开发稳定的丙二酰硫酯电子等排体， 阐明分子相互作用和构象变化，导致有效的催化。的目的 本发明的建议是克服与天然丙二酰硫代酯和先前合成的丙二酰硫代酯相关的问题， 等排体。我们有一组丙二酰硫酯，它们保留了在先前的电子等排体中丢失的关键酮。我们 第一个目标是解决晶体或cryo-EM结构的酰基辅酶A羧化酶在复杂的与我们最好的 电子等排体来阐明酶：底物相互作用和构象变化。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-EM或NMR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 丙二酰硫酯类似物与羧酸盐电子等排体在捕获酶：底物相互作用中的用途。我们 结构将揭示催化过程中的构象变化，可以作为药物设计的目标， 在酶工程中被计算在内。

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.