title: A Competitive Enzyme-Linked Immunosorbent Assay System for Adenylation Domains in Nonribosomal Peptide Synthetases.
abstract: We describe a proof-of-concept study of a competitive enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) with active-site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold. A biotin functionality immobilizes the probes onto a streptavidin-coated solid support. Dissociation constants were determined with a series of ligands, including enzyme substrates and a library of sulfamoyloxy-linked aminoacyl/aryl-AMP analogues. As it enables direct readout of protein-ligand interaction, the competitive ELISA technique provided information on comparative structure- activity relationships and insights into the enzyme active-site architecture of NRPS A-domains. These studies indicate that the ELISA technique can accelerate the discovery of small-molecule inhibitors of the A-domains with new scaffolds that perturb the production of NRPS-related virulence factors.
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: 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: D000249:Adenosine Monophosphate / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D010453:Peptide Synthases / Q000737:chemistry*
chemical_list: D000249:Adenosine Monophosphate; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: active site-directed probe; adenylation domain; biosynthesis; enzyme-linked immunosorbent assay; enzymes