Active site-directed proteomic probes for adenylation domains in nonribosomal peptide synthetases.
Active site-directed proteomic probes for adenylation domains in nonribosomal peptide synthetases.
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DOI：
10.1039/c4cc09412c
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发表时间：
2015-02-11
期刊：
影响因子：
--
通讯作者：
Kakeya H
中科院分区：
文献类型：
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作者：
Konno S;Ishikawa F;Suzuki T;Dohmae N;Burkart MD;Kakeya H
在所有非核糖体肽合成酶(NRPS)模块中发现的腺基化(A)结构域是必需的催化成分，在非核糖体肽生物合成过程中充当选择氨基酸构建块的守门人。利用这些酶的严格底物识别特性，我们瞄准了针对NRPS中A结构域的活性位点定向蛋白质组探针的开发，从而能够检测、分离、鉴定和鉴定天然蛋白质组中的A结构域。在这里，我们描述了一种选择性化学标记NRPS酶中单个A结构域的一般策略，使用活性位点定向的蛋白质组探针连接到具有可点击的二苯甲酮功能的5‘-O-N-氨基酰基腺苷(AMS)支架上。这些探针通过配体导向的蛋白质标记选择性地针对天然产物生产者蛋白质组中的单个A结构域。这些数据表明，这些蛋白质组学工具可以极大地促进复杂生物系统中NRPS酶的几乎任何一种A结构域的分子鉴定、功能表征和图谱分析。
Adenylation (A) domains found in all nonribosomal peptide synthetase (NRPS) modules are essential catalytic components and function as gatekeepers to select amino acid building blocks during nonribosomal peptide biosynthesis. Leveraging the strict substrate recognition characteristics of these enzymes, we targeted the development of active site-directed proteomic probes for A domains in NRPSs that enable detection, isolation, identification, and enzymatic characterization of A domains in native proteomes. Here, we describe a general strategy for selective chemical labeling of individual A domains in NRPS enzymes using active site-directed proteomic probes coupled to 5′-O-N-(aminoacyl)sulfamoyladenosine (AMS) scaffold with a clickable benzophenone functionality. These probes selectively target individual A domains in natural product producer proteomes by ligand-directed protein labeling. The data demonstrate that these proteomic tools can greatly facilitate the molecular identification, functional characterization, and profiling of virtually any kind of A domains of NRPS enzymes in complex biological systems.