title: Measurement of Nonribosomal Peptide Synthetase Adenylation Domain Activity Using a Continuous Hydroxylamine Release Assay.
abstract: Adenylation is a crucial enzymatic process in the biosynthesis of nonribosomal peptide synthetase (NRPS) derived natural products. Adenylation domains are considered the gatekeepers of NRPSs since they select, activate, and load the carboxylic acid substrate onto a downstream peptidyl carrier protein (PCP) domain of the NRPS. We describe a coupled continuous kinetic assay for NRPS adenylation domains that substitutes the PCP domain with hydroxylamine as the acceptor molecule. The pyrophosphate released from the first-half reaction is then measured using a two-enzyme coupling system, which detects conversion of the chromogenic substrate 7-methylthioguanosine (MesG) to 7-methylthioguanine. From profiling substrate specificity of unknown or engineered adenylation domains to studying chemical inhibition of adenylating enzymes, this robust assay will be of widespread utility in the broad field NRPS enzymology.
authors.affiliation: Department of Medicinal Chemistry, University of Minnesota, 8-101 Weaver-Densford Hall, 308 Harvard St. SE, Minneapolis, MN, 55455, USA.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Duckworth
authors.affiliation: Center for Drug Design, University of Minnesota, Minneapolis, MN, 55455, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Wilson
authors.affiliation: Department of Medicinal Chemistry, University of Minnesota, 8-101 Weaver-Densford Hall, 308 Harvard St. SE, Minneapolis, MN, 55455, USA.
authors.forename: Courtney C
authors.initials: CC
authors.lastname: Aldrich
mesh_terms: D001419:Bacteria / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D006151:Guanosine / Q000031:analogs & derivatives / Q000378:metabolism; D019811:Hydroxylamine / Q000378:metabolism*; D010453:Peptide Synthases / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D006151:Guanosine; D019811:Hydroxylamine; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: Adenylate-forming; Adenylation; Enzyme assay; Hydroxamate; MesG
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