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

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 LOPAC(1280) library.

SUBMITTER: Wilson DJ

PROVIDER: S-EPMC3135573 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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

Wilson Daniel J DJ Shi Ce C Duckworth Benjamin P BP Muretta Joseph M JM Manjunatha Ujjini U Sham Yuk Y YY Thomas David D DD Aldrich Courtney C CC

Analytical biochemistry 20110508 1

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 measur ...[more]

PMID: 21621502

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