title: A Multiple-Labeling Strategy for Nonribosomal Peptide Synthetases Using Active-Site-Directed Proteomic Probes for Adenylation Domains.
abstract: 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.
authors.affiliation: 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. fishika@pharm.kyoto-u.ac.jp.
authors.forename: Fumihiro
authors.initials: F
authors.lastname: Ishikawa
authors.affiliation: Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
authors.forename: Takehiro
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
authors.forename: Naoshi
authors.initials: N
authors.lastname: Dohmae
authors.affiliation: 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. scseigyo-hisyo@pharm.kyoto-u.ac.jp.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Kakeya
mesh_terms: D001386:Azides / Q000737:chemistry; D056449:Bacillales / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D057930:Click Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015335:Molecular Probes / Q000737:chemistry*; D010453:Peptide Synthases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D020543:Proteome / Q000032:analysis
chemical_list: D001386:Azides; D001426:Bacterial Proteins; D015335:Molecular Probes; D020543:Proteome; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: active site-directed proteomic probe; adenylation domain; nonribosomal peptide synthetase; photoaffinity labelling; proteomics