Profiling Nonribosomal Peptide Synthetase Activities Using Chemical Proteomic Probes for Adenylation Domains
Profiling Nonribosomal Peptide Synthetase Activities Using Chemical Proteomic Probes for Adenylation Domains
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DOI：
10.1021/acschembio.5b00097
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发表时间：
2015-09-01
影响因子：
4
通讯作者：
Kakeya, Hideaki
中科院分区：
文献类型：
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作者：
Ishikawa, Fumihiro;Konno, Sho;Kakeya, Hideaki
非核糖体肽合成酶 (NRPS) 和聚酮化合物合成酶是生物合成酶的一个大家族，可催化具有重要生物学活性的天然产物的合成。遗传学研究极大地促进了我们对这些生物合成酶的理解。然而，蛋白质组学研究是有限的。在这里，我们描述了腺苷酸化 (A) 域的活性定点蛋白质组探针的应用，以直接在天然蛋白质组环境中分析 NRPS 的活性。附加可点击二苯甲酮功能的 5'-O-N-（氨酰基）氨磺酰基腺苷的衍生化能够对蛋白质组提取物中 NRPS 中的 A 结构域进行基于活性的蛋白质分析。这些探针用于鉴定产生天然产物的微生物、优化培养条件并分析 NRPS 的活性动态。我们的蛋白质组学方法提供了一种简单且通用的方法来监测蛋白质水平上的 NRPS 表达，并将有助于鉴定参与次级代谢产物生产的孤儿酶途径。
Nonribosomal peptide synthetases (NRPSs) and polyketide synthases are large diverse families of biosynthetic enzymes that catalyze the synthesis of natural products that display biologically important activities. Genetic investigations have greatly contributed to our understanding of these biosynthetic enzymes; however, proteomic studies are limited. Here we describe the application of active site-directed proteomic probes for adenylation (A) domains to profile the activity of NRPSs directly in native proteomic environments. Derivatization of a 5'-O-N-(aminoacyl)sulfamoyladenosine appended clickable benzophenone functionality enabled activity-based protein profiling of the A-domains in NRPSs in proteomic extracts. These probes were used to identify natural product producing microorganisms, optimize culture conditions, and profile the activity dynamics of NRPSs. Our proteomic approach offers a simple and versatile method to monitor NRPS expression at the protein level and will facilitate the identification of orphan enzymatic pathways involved in secondary metabolite production.