title: Profiling Nonribosomal Peptide Synthetase Activities Using Chemical Proteomic Probes for Adenylation Domains.
abstract: 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.
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.
authors.forename: Fumihiro
authors.initials: F
authors.lastname: Ishikawa
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.
authors.forename: Sho
authors.initials: S
authors.lastname: Konno
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.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Kakeya
mesh_terms: D001419:Bacteria / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D020134:Catalytic Domain; D015335:Molecular Probes / Q000737:chemistry / Q000378:metabolism*; D010453:Peptide Synthases / Q000737:chemistry / Q000378:metabolism*; D048630:Polyketide Synthases / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods
chemical_list: D015335:Molecular Probes; D048630:Polyketide Synthases; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase