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. 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. The robust nature and simplicity of the reaction protocol offers advantages over existing methods for ATP quantification and NRPS substrate analysis.

中文翻译：

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靛蓝合成酶 BpsA 提供了一种比色 ATP 测定，可用于量化其他 NRPS 酶的底物偏好

开发基于蓝色色素合成非核糖体肽合成酶 (NRPS) BpsA 的 ATP 比色分析，并证明其在定义其他 NRPS 酶的底物特异性方面的效用。BpsA 能够将两分子 L-谷氨酰胺转化为易于检测的蓝色颜料靛蓝，在此过程中消耗两分子 ATP。我们表明该反应的化学计量是可靠的，并且它可以在微孔板格式中进行，以使用分光光度计读板器将各种介质中的 ATP 浓度准确量化到低微摩尔水平。我们还证明，通过添加焦磷酸酶来驱动 ATP 在首选底物存在下的消耗，该测定可适用于评估 NRPS 腺苷酸化域的氨基酸底物偏好。