Abstract
Objectives
To develop a colorimetric assay for ATP based on the blue-pigment synthesising non-ribosomal peptide synthetase (NRPS) BpsA, and to demonstrate its utility in defining the substrate specificity of other NRPS enzymes.
Results
BpsA is able to convert two molecules of L-glutamine into the readily-detected blue pigment indigoidine, consuming two molecules of ATP in the process. We showed that the stoichiometry of this reaction is robust and that it can be performed in a microplate format to accurately quantify ATP concentrations to low micromolar levels in a variety of media, using a spectrophotometric plate-reader. We also demonstrated that the assay can be adapted to evaluate the amino acid substrate preferences of NRPS adenylation domains, by adding pyrophosphatase enzyme to drive consumption of ATP in the presence of the preferred substrate.
Conclusions
The robust nature and simplicity of the reaction protocol offers advantages over existing methods for ATP quantification and NRPS substrate analysis.
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Acknowledgements
This work was funded by the Health Research Council of New Zealand (contract 16/172 awarded to DFA and JGO) and was inspired by research previously funded by the Royal Society of New Zealand Marsden Fund (contract VUW0901 awarded to DFA). JGO is supported by a Royal Society of New Zealand Rutherford Discovery Fellowship (contract RDF-VUW1601), MJC by a Royal Society of New Zealand Marsden Fast Start Grant (contract 18-VUW-082) and VMC by a Victoria University of Wellington Masters scholarship.
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Brown, A.S., Calcott, M.J., Collins, V.M. et al. The indigoidine synthetase BpsA provides a colorimetric ATP assay that can be adapted to quantify the substrate preferences of other NRPS enzymes. Biotechnol Lett 42, 2665–2671 (2020). https://doi.org/10.1007/s10529-020-02972-4
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DOI: https://doi.org/10.1007/s10529-020-02972-4