Colorimetric Determination of Adenylation Domain Activity in Nonribosomal Peptide Synthetases by Using Chrome Azurol S

Adenylation domains are the main contributor to structural complexity among nonribosomal peptides due to their varied but stringent substrate selection. Several in vitro assays to determine the substrate specificity of these dedicated biocatalysts have been implemented, but high sensitivity is often...

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Bibliographic Details
Published in:Chembiochem : a European journal of chemical biology 2023-03, Vol.24 (5), p.e202200668-n/a
Main Authors: Kahlert, Lukas, Lichstrahl, Michael S., Townsend, Craig A.
Format: Article
Language:English
Subjects:
Adenylation
adenylation domains
amino acids
Biocatalysts
chromophores
colorimetric assays
Colorimetry
Colorimetry - methods
Complexity
Iron
Peptide Synthases - metabolism
Peptides
pyrophosphates
Reagents
Substrate Specificity
Substrates
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Summary:Adenylation domains are the main contributor to structural complexity among nonribosomal peptides due to their varied but stringent substrate selection. Several in vitro assays to determine the substrate specificity of these dedicated biocatalysts have been implemented, but high sensitivity is often accompanied by the cost of laborious procedures, expensive reagents or the requirement for auxiliary enzymes. Here, we describe a simple protocol that is based on the removal of ferric iron from a preformed chromogenic complex between ferric iron and Chrome Azurol S. Adenylation activity can be rapidly followed by a decrease in absorbance at 630 nm, visualized by a prominent color change from blue to orange. Stealing iron: The chromogenic complex between Chrome Azurol S and ferric iron was repurposed to monitor the substrate specificity of adenylation domains in vitro. This assay avoids the requirement for expensive co‐substrates or auxiliary enzymes and can also be applied for adenylation domains that are embedded in entire modules.
ISSN:1439-4227
1439-7633
1439-7633
DOI:10.1002/cbic.202200668