Measurement of nonribosomal peptide synthetase adenylation domain activity using a continuous hydroxylamine release assay

Benjamin P. Duckworth; Daniel J. Wilson; Courtney C. Aldrich

doi:10.1007/978-1-4939-3375-4_3

Measurement of nonribosomal peptide synthetase adenylation domain activity using a continuous hydroxylamine release assay

Benjamin P. Duckworth, Daniel J. Wilson, Courtney C. Aldrich

Research output: Chapter in Book/Report/Conference proceeding › Chapter

7 Scopus citations

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.

Original language	English (US)
Title of host publication	Methods in Molecular Biology
Publisher	Humana Press Inc.
Pages	53-61
Number of pages	9
DOIs	https://doi.org/10.1007/978-1-4939-3375-4_3
State	Published - Feb 1 2016

Publication series

Name	Methods in Molecular Biology
Volume	1401
ISSN (Print)	1064-3745

Bibliographical note

Funding Information:
C.C.A. acknowledges membership in and support from the Region V ?Great Lakes? Regional Center of Excellence in Biodefense and Emerging Infectious Diseases Consortium (National Institutes of Health award 1-U54-AI-057153) and support from R01-AI-070219 for this work.

Keywords

Adenylate-forming
Adenylation
Enzyme assay
Hydroxamate
MesG

Access

10.1007/978-1-4939-3375-4_3

http://europepmc.org/articles/pmc5588023

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Cite this

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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.",

keywords = "Adenylate-forming, Adenylation, Enzyme assay, Hydroxamate, MesG",

author = "Duckworth, {Benjamin P.} and Wilson, {Daniel J.} and Aldrich, {Courtney C.}",

note = "Funding Information: C.C.A. acknowledges membership in and support from the Region V ?Great Lakes? Regional Center of Excellence in Biodefense and Emerging Infectious Diseases Consortium (National Institutes of Health award 1-U54-AI-057153) and support from R01-AI-070219 for this work.",

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AU - Wilson, Daniel J.

AU - Aldrich, Courtney C.

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N2 - 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.

AB - 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.

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