The indigoidine synthetase BpsA provides a colorimetric ATP assay that can be adapted to quantify the substrate preferences of other NRPS enzymes.

IF 2 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biotechnology Letters Pub Date : 2020-12-01 Epub Date: 2020-07-17 DOI:10.1007/s10529-020-02972-4

Alistair S Brown, Mark J Calcott, Vincent M Collins, Jeremy G Owen, David F Ackerley

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引用次数: 6

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

目的:建立一种基于蓝色色素合成非核糖体肽合成酶(NRPS) BpsA的ATP比色测定方法，并证明其在确定其他NRPS酶的底物特异性方面的实用性。结果:BpsA能够将两分子l -谷氨酰胺转化为易于检测的蓝色色素靛蓝素，在此过程中消耗两分子ATP。我们表明，该反应的化学计量学是稳健的，并且可以在微孔板格式中进行，使用分光光度板阅读器，在各种介质中准确地将ATP浓度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在首选底物存在的情况下ATP的消耗，该分析可以适用于评估NRPS腺苷化结构域的氨基酸底物偏好。结论:与现有的ATP定量和NRPS底物分析方法相比，该反应方案具有鲁棒性和简单性。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Biotechnology Letters 工程技术-生物工程与应用微生物

CiteScore

5.90

自引率

3.70%

发文量

108

审稿时长

1.2 months

期刊介绍： Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them. All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included. Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields. The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories. Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.