使用连续羟胺释放测定法测量非核糖体肽合成酶腺苷化结构域活性
Measurement of Nonribosomal Peptide Synthetase Adenylation Domain Activity Using a Continuous Hydroxylamine Release Assay.
作者信息
Duckworth Benjamin P, Wilson Daniel J, Aldrich Courtney C
机构信息
Department of Medicinal Chemistry, University of Minnesota, 8-101 Weaver-Densford Hall, 308 Harvard St. SE, Minneapolis, MN, 55455, USA.
Center for Drug Design, University of Minnesota, Minneapolis, MN, 55455, USA.
出版信息
Methods Mol Biol. 2016;1401:53-61. doi: 10.1007/978-1-4939-3375-4_3.
Abstract
Adenylation is a crucial enzymatic process in the biosynthesis of nonribosomal peptide synthetase (NRPS) derived natural products. Adenylation domains are considered the gatekeepers of NRPSs since they select, activate, and load the carboxylic acid substrate onto a downstream peptidyl carrier protein (PCP) domain of the NRPS. We describe a coupled continuous kinetic assay for NRPS adenylation domains that substitutes the PCP domain with hydroxylamine as the acceptor molecule. The pyrophosphate released from the first-half reaction is then measured using a two-enzyme coupling system, which detects conversion of the chromogenic substrate 7-methylthioguanosine (MesG) to 7-methylthioguanine. From profiling substrate specificity of unknown or engineered adenylation domains to studying chemical inhibition of adenylating enzymes, this robust assay will be of widespread utility in the broad field NRPS enzymology.
摘要
腺苷化是源自非核糖体肽合成酶（NRPS）的天然产物生物合成中的关键酶促过程。腺苷化结构域被认为是NRPS的守门人，因为它们选择、激活羧酸底物并将其加载到NRPS的下游肽基载体蛋白（PCP）结构域上。我们描述了一种用于NRPS腺苷化结构域的耦合连续动力学测定法，该方法用羟胺作为受体分子替代PCP结构域。然后使用双酶偶联系统测量上半反应释放的焦磷酸，该系统检测生色底物7-甲基硫代鸟苷（MesG）向7-甲基硫代鸟嘌呤的转化。从分析未知或工程化腺苷化结构域的底物特异性到研究腺苷化酶的化学抑制作用，这种强大的测定法将在广泛的NRPS酶学领域中具有广泛的用途。
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