| Chapter title |
Colorimetric Detection of the Adenylation Activity in Nonribosomal Peptide Synthetases
|
|---|---|
| Chapter number | 5 |
| Book title |
Nonribosomal Peptide and Polyketide Biosynthesis
|
| Published in |
Methods in molecular biology, January 2016
|
| DOI | 10.1007/978-1-4939-3375-4_5 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-3373-0, 978-1-4939-3375-4
|
| Authors |
Chitose Maruyama, Haruka Niikura, Masahiro Takakuwa, Hajime Katano, Yoshimitsu Hamano, Maruyama, Chitose, Niikura, Haruka, Takakuwa, Masahiro, Katano, Hajime, Hamano, Yoshimitsu |
| Abstract |
Nonribosomal peptide synthetases (NRPSs) are multifunctional enzymes consisting of catalytic domains. The substrate specificities of adenylation (A) domains determine the amino-acid building blocks to be incorporated during nonribosomal peptide biosynthesis. The A-domains mediate ATP-dependent activation of amino-acid substrates as aminoacyl-O-AMP with pyrophosphate (PPi) release. Traditionally, the enzymatic activity of the A-domains has been measured by radioactive ATP-[(32)P]-PPi exchange assays with the detection of (32)P-labeled ATP. Recently, we developed a colorimetric assay for the direct detection of PPi as a yellow 18-molybdopyrophosphate anion ([(P2O7)Mo18O54](4-)). [(P2O7)Mo18O54](4-) was further reduced by ascorbic acid to give a more readily distinguishable blue coloration. Here we demonstrate the lab protocols for the colorimetric assay of PPi released in A-domain reactions. |
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