Ishikawa Fumihiro, Suzuki Takehiro, Dohmae Naoshi, Kakeya Hideaki
Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto, 606-8501, Japan.
Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
Chembiochem. 2015 Dec;16(18):2590-4. doi: 10.1002/cbic.201500481. Epub 2015 Nov 6.
Genetic approaches have greatly contributed to our understanding of nonribosomal peptide biosynthetic machinery; however, proteomic investigations are limited. Here, we developed a highly sensitive detection strategy for multidomain nonribosomal peptide synthetases (NRPSs) by using a multiple-labeling technique with active-site-directed probes for adenylation domains. When applied to gramicidin S-producing and -nonproducing strains of Aneurinibacillus migulanus (DSM 5759 and DSM 2895, respectively), the multiple technique sensitively detected an active multidomain NRPS (GrsB) in lysates obtained from the organisms. This functional proteomics method revealed an unknown inactive precursor (or other inactive form) of GrsB in the nonproducing strain. This method provides a new option for the direct detection, functional analysis, and high-resolution identification of low-abundance active NRPS enzymes in native proteomic environments.
遗传学方法极大地促进了我们对非核糖体肽生物合成机制的理解；然而，蛋白质组学研究却很有限。在此，我们通过使用针对腺苷化结构域的活性位点导向探针的多重标记技术，开发了一种用于多结构域非核糖体肽合成酶（NRPSs）的高灵敏度检测策略。当应用于产短杆菌肽S和不产短杆菌肽S的迁移类芽孢杆菌菌株（分别为DSM 5759和DSM 2895）时，该多重技术灵敏地检测到了从这些生物体获得的裂解物中的一种活性多结构域NRPS（GrsB）。这种功能蛋白质组学方法揭示了不产菌株中GrsB的一种未知无活性前体（或其他无活性形式）。该方法为在天然蛋白质组环境中直接检测、功能分析和高分辨率鉴定低丰度活性NRPS酶提供了一种新选择。
