title: Affinity Purification Method for the Identification of Nonribosomal Peptide Biosynthetic Enzymes Using a Synthetic Probe for Adenylation Domains.
abstract: A series of inhibitors have been designed based on 5'-O-sulfamoyl adenosine (AMS) that display tight binding characteristics towards the inhibition of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs). We recently developed an affinity probe for A domains that could be used to facilitate the specific isolation and identification of NRPS modules. Our synthetic probe, which is a biotinylated variant of L-Phe-AMS (L-Phe-AMS-biotin), selectively targets the A domains in NRPS modules that recognize and convert L-Phe to an aminoacyl adenylate in whole proteomes. In this chapter, we describe the design and synthesis of L-Phe-AMS-biotin and provide a summary of our work towards the development of a series of protocols for the specific enrichment of NRPS modules using this probe.
authors.affiliation: Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Science, Kyoto University, Sakyo-ku, 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 Science, Kyoto University, Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Kakeya
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D000591:Amino Acid Isomerases / Q000737:chemistry / Q000302:isolation & purification*; D056449:Bacillales / Q000737:chemistry / Q000201:enzymology*; D001710:Biotin / Q000138:chemical synthesis / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D010453:Peptide Synthases / Q000737:chemistry / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary
chemical_list: C003360:5'-sulfamoyladenosine; D001710:Biotin; D000591:Amino Acid Isomerases; C022343:phenylalanine racemase (ATP-hydrolyzing); D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; D000241:Adenosine
keywords: Adenylation domain; Affinity purification; Aneurinibacillus migulanus ATCC 9999; Gramicidin S synthase 1 (GrsA); Nonribosomal peptide synthetase (NRPS); Small-molecule probe