Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0358, USA.
J Am Chem Soc. 2012 Jan 18;134(2):769-72. doi: 10.1021/ja2082334. Epub 2011 Dec 29.
We targeted the development of a dehydratase (DH)-specific reactive probe that can facilitate detection, enrichment, and identification of DH enzymes in fatty acid synthases (FASs) and polyketide synthases (PKSs). The first reported mechanism-based inactivator, 3-decynoyl-N-acetylcysteamine (3-decynoyl-NAC), while active against the Escherichia coli β-hydroxydecanoyl thiol ester DH FabA, translates poorly to an activity-based probe because of nonspecific reactivity of the thioester moiety. Here we describe the design, synthesis, and utility of a DH-specific probe that contains a sulfonyl 3-alkyne reactive warhead engineered to avoid hydrolysis or nonenzymatic inactivation. When coupled with a fluorescent tag, this probe targets DH enzymes from recombinant type I and type II FAS and PKS enzyme systems and in whole proteomes. Activity studies, including FabA inactivation and antibiotic susceptibility, suggest that this sulfonyl 3-alkyne scaffold selectively targets a common DH mechanism. These studies indicate that the DH-specific mechanism-based probe can greatly accelerate both the functional characterization and molecular identification of virtually any type of FAS and PKS in complex proteomes.
我们的目标是开发一种脱水酶（DH）特异性反应探针，以促进脂肪酸合酶（FAS）和聚酮合酶（PKS）中 DH 酶的检测、富集和鉴定。第一个报道的基于机制的失活剂 3-癸炔酰基-N-乙酰半胱氨酸胺（3-decynoyl-NAC）虽然对大肠杆菌β-羟癸酰硫酯 DH FabA 有效，但由于硫酯部分的非特异性反应，其转化为活性探针的效果不佳。在这里，我们描述了一种 DH 特异性探针的设计、合成和应用，该探针包含一个磺酰基 3-炔基反应弹头，旨在避免水解或非酶失活。当与荧光标记物结合时，该探针可靶向来自重组 I 型和 II 型 FAS 和 PKS 酶系统以及整个蛋白质组的 DH 酶。活性研究，包括 FabA 失活和抗生素敏感性，表明这个磺酰基 3-炔基支架选择性地针对一个常见的 DH 机制。这些研究表明，DH 特异性的基于机制的探针可以极大地加速复杂蛋白质组中几乎任何类型的 FAS 和 PKS 的功能表征和分子鉴定。
