Cofactor biosynthetic pathways represent a rich source of potential antibiotic targets. The second step in biotin biosynthesis is performed by BioA, a pyridoxal 5′-phosphate (PLP)-dependent enzyme. This enzyme has been confirmed as a candidate target in Mycobacterium tuberculosis; however, the current bioassay used to measure BioA activity is cumbersome and low throughput. Here we describe the design, development, and optimization of a continuous coupled fluorescence displacement assay to measure BioA activity. In this coupled assay, BioD converts the product of the BioA-catalyzed reaction into dethiobiotin, which is subsequently detected by displacement of a fluorescently labeled dethiobiotin probe from streptavidin. The assay was further adapted to a high-throughput screening format and validated against the LOPAC1280 library.
Bibliographical noteFunding Information:
This work was supported in part by the Bill and Melinda Gates Foundation and Wellcome Trust for the Grand Challenges in Global Health project “Drugs for Treatment of Latent Tuberculosis” and by National Institutes of Health (NIH) grants AI-091790 (C.C.A.), GM-27906 (D.D.T.), and AR-056191 (J.M.M.).
- Fluorescence displacement assay
- High-throughput screening
- Mycobacterium tuberculosis