Sulfonyl 3-alkynyl pantetheinamides as mechanism-based cross-linkers of acyl carrier protein dehydratase.
Sulfonyl 3-alkynyl pantetheinamides as mechanism-based cross-linkers of acyl carrier protein dehydratase.
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DOI：
10.1021/ja4042059
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发表时间：
2013-06-19
影响因子：
15
通讯作者：
Burkart, Michael D.
中科院分区：
文献类型：
--
作者：
Ishikawa, Fumihiro;Haushalter, Robert W.;Lee, D. John;Finzel, Kara;Burkart, Michael D.
酰基载体蛋白（ACP）在乙酸生物合成途径中起着核心作用，作为底物和生长中间体的系链。活性和结构研究强调了这种作用的复杂性，其蛋白质-蛋白质相互作用最近作为催化调节剂受到审查。由于现有的方法来询问这些相互作用已经不足，我们试图开发新的工具来帮助他们的研究。在这里，我们描述了泛酰巯基乙胺酰胺的设计，合成和应用，能够交联ACP与催化β-羟酰基载体蛋白酶（DH）结构域的基础上的3-炔基砜弹头。我们证明了这一过程中的应用，大肠杆菌脂肪酸合酶，并将其应用于探针蛋白质与非同源载体蛋白的相互作用。最后，我们使用溶液相蛋白质NMR来证明磺酰基-3-炔基泛酰巯基乙胺酰胺被ACP完全螯合，这表明隐-ACP紧密地模仿天然DH底物。这种交联技术通过提供机械交联酶复合物的途径，提供了将这些生物合成酶锁定在其天然结合状态的直接潜力，为持续的结构挑战提供了解决方案。
The acyl carrier protein (ACP) plays a central function in acetate biosynthetic pathways, serving as a tether for substrates and growing intermediates. Activity and structural studies have highlighted the complexities of this role, and its protein-protein interactions have recently come under scrutiny as a regulator of catalysis. As existing methods to interrogate these interactions have fallen short, we have sought to develop new tools to aid their study. Here we describe the design, synthesis, and application of pantetheinamides capable of crosslinking ACPs with catalytic β-hydroxyacyl carrier protein dehydratase (DH) domains based upon a 3-alkynyl sulfone warhead. We demonstrate this process by application to the Escherichia coli fatty acid synthase and apply it to probe protein-protein interactions with non-cognate carrier proteins. Finally, we use solution phase protein NMR to demonstrate that sulfonyl-3-alkynyl pantetheinamide is fully sequestered by the ACP, indicating that the crypto-ACP closely mimics the natural DH substrate. This crosslinking technology offers immediate potential to lock these biosynthetic enzymes in their native binding states by providing access to mechanistically-crosslinked enzyme complexes, presenting a solution to ongoing structural challenges.
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DOI：
10.1016/s0969-2126(00)00178-7
发表时间：
2000-08-15
期刊：
STRUCTURE WITH FOLDING & DESIGN
影响因子：
--
作者：
Parris, KD;Lin, L;Somers, WS
通讯作者：
Somers, WS
影响因子：
2.9
作者：
Mitchell CA;Shi C;Aldrich CC;Gulick AM
通讯作者：
Gulick AM
影响因子：
--
作者：
Liu Y;Zheng T;Bruner SD
通讯作者：
Bruner SD
影响因子：
--
作者：
Ploskon, Eliza;Arthur, Christopher J.;Crump, Matthew P.
通讯作者：
Crump, Matthew P.
影响因子：
--
作者：
La Clair, JJ;Foley, TL;Burkart, MD
通讯作者：
Burkart, MD