| Chapter title |
Measurement of Nonribosomal Peptide Synthetase Adenylation Domain Activity Using a Continuous Hydroxylamine Release Assay
|
|---|---|
| Chapter number | 3 |
| Book title |
Nonribosomal Peptide and Polyketide Biosynthesis
|
| Published in |
Methods in molecular biology, January 2016
|
| DOI | 10.1007/978-1-4939-3375-4_3 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-3373-0, 978-1-4939-3375-4
|
| Authors |
Benjamin P. Duckworth, Daniel J. Wilson, Courtney C. Aldrich, Duckworth, Benjamin P., Wilson, Daniel J., Aldrich, Courtney C. |
| 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. |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Germany | 1 | 3% |
| Unknown | 39 | 98% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Researcher | 7 | 18% |
| Student > Ph. D. Student | 6 | 15% |
| Student > Master | 4 | 10% |
| Student > Bachelor | 3 | 8% |
| Student > Postgraduate | 2 | 5% |
| Other | 5 | 13% |
| Unknown | 13 | 33% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 12 | 30% |
| Chemistry | 7 | 18% |
| Computer Science | 2 | 5% |
| Agricultural and Biological Sciences | 2 | 5% |
| Medicine and Dentistry | 1 | 3% |
| Other | 1 | 3% |
| Unknown | 15 | 38% |