title: Specific enrichment of nonribosomal peptide synthetase module by an affinity probe for adenylation domains.
abstract: We targeted the development of an affinity probe for adenylation (A) domains that can facilitate enrichment, identification, and quantification of A domain-containing modules in nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) hybrids and NRPSs. A 5'-O-sulfamoyladenosine (AMS) non-hydrolyzable analogue of adenosine monophosphate (AMP) has been reported as a scaffold for the design of inhibitors exhibiting tight binding of adenylation enzymes. Here we describe the application of an affinity probe for A domains. Our synthetic probe, a biotinylated L-Phe-AMS (L-Phe-AMS-biotin) specifically targets the A domains in NRPS modules that activates L-Phe to an aminoacyladenylate intermediate in both recombinant NRPS enzyme systems and whole proteomes.
authors.affiliation: Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. Electronic address: fishika@pharm.kyoto-u.ac.jp.
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-ku, Kyoto 606-8501, Japan. Electronic address: scseigyo-hisyo@pharm.kyoto-u.ac.jp.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Kakeya
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry*; D000595:Amino Acid Sequence; D015195:Drug Design; D020128:Inhibitory Concentration 50; D015335:Molecular Probes / Q000737:chemistry*; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D040901:Proteomics; D012270:Ribosomes / Q000201:enzymology
chemical_list: D015335:Molecular Probes; D000249:Adenosine Monophosphate; D010453:Peptide Synthases
keywords: Adenylation domain; Affinity probe; Gramicidin S; Nonribosomal peptide synthetase; Polyketide synthase