Measurement of Nonribosomal Peptide Synthetase Adenylation Domain Activity Using a Continuous Hydroxylamine Release Assay

Methods in Molecular Biology
Benjamin P DuckworthCourtney C Aldrich

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.

Citations

Apr 1, 2018·Chembiochem : a European Journal of Chemical Biology·Jesus F BarajasJay D Keasling
Mar 12, 2019·Chembiochem : a European Journal of Chemical Biology·Serina L RobinsonLawrence P Wackett
Sep 14, 2019·Biotechnology Advances·Antoine VassauxValérie Leclère
Dec 12, 2018·Biochemistry·Trey A RonnebaumAudrey L Lamb

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