A continuous fluorescence displacement assay for BioA: An enzyme involved in biotin biosynthesis

Daniel J. Wilson, Ce Shi, Benjamin P. Duckworth, Joseph M. Muretta, Ujjini Manjunatha, Yuk Y. Sham, David D. Thomas, Courtney C. Aldrich

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)27-38
Number of pages12
JournalAnalytical Biochemistry
Volume416
Issue number1
DOIs
StatePublished - Sep 1 2011

Bibliographical note

Funding 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.).

Keywords

  • Biotin
  • Dethiobiotin
  • Fluorescence displacement assay
  • High-throughput screening
  • Mycobacterium tuberculosis

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