A Competitive Enzyme‐Linked Immunosorbent Assay System for Adenylation Domains in Nonribosomal Peptide Synthetases. (16th February 2016)

• Record Type: Journal Article
• Authors: Ishikawa, Fumihiro; Kakeya, Hideaki
• Is Part Of: Chembiochem. Volume 17:Number 6(2016)
• Page Start: 474
• Page End: 478
• Publication Date: 2016-02-16
• Subjects: active site-directed probe -- adenylation domain -- biosynthesis -- enzyme-linked immunosorbent assay -- enzymes; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201500553
• Languages: English
• ISSNs: 1439-4227

Abstract

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. Abstract : ELISA for enzyme active‐site insights : A competitive ELISA system for adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) uses active‐site‐directed probes. This technique was used to provide data on the structure–activity relationships and enzyme active‐site architecture of the A‐domains of NRPS enzymes.