Ishikawa Fumihiro, Kakeya Hideaki
Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Science, Kyoto University, Sakyo-ku, Kyoto, 606-8501, Japan.
Methods Mol Biol. 2016;1401:63-76. doi: 10.1007/978-1-4939-3375-4_4.
A series of inhibitors have been designed based on 5'-O-sulfamoyl adenosine (AMS) that display tight binding characteristics towards the inhibition of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs). We recently developed an affinity probe for A domains that could be used to facilitate the specific isolation and identification of NRPS modules. Our synthetic probe, which is a biotinylated variant of L-Phe-AMS (L-Phe-AMS-biotin), selectively targets the A domains in NRPS modules that recognize and convert L-Phe to an aminoacyl adenylate in whole proteomes. In this chapter, we describe the design and synthesis of L-Phe-AMS-biotin and provide a summary of our work towards the development of a series of protocols for the specific enrichment of NRPS modules using this probe.
基于5'-O-氨磺酰腺苷（AMS）设计了一系列抑制剂，这些抑制剂对非核糖体肽合成酶（NRPSs）中的腺苷化（A）结构域具有紧密结合特性，可抑制其活性。我们最近开发了一种针对A结构域的亲和探针，可用于促进NRPS模块的特异性分离和鉴定。我们的合成探针是L-苯丙氨酸-AMS（L-Phe-AMS）的生物素化变体（L-Phe-AMS-生物素），它能选择性地靶向NRPS模块中的A结构域，这些A结构域在完整蛋白质组中识别L-苯丙氨酸并将其转化为氨酰腺苷酸。在本章中，我们描述了L-Phe-AMS-生物素的设计与合成，并总结了我们利用该探针开发一系列特异性富集NRPS模块方案的工作。
