title: 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.
authors.affiliation: Center for Drug Design, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Wilson
authors.forename: Ce
authors.initials: C
authors.lastname: Shi
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Duckworth
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Muretta
authors.forename: Ujjini
authors.initials: U
authors.lastname: Manjunatha
authors.forename: Yuk Y
authors.initials: YY
authors.lastname: Sham
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
authors.forename: Courtney C
authors.initials: CC
authors.lastname: Aldrich
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D001710:Biotin / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000138:chemical synthesis / Q000737:chemistry; D018360:Crystallography, X-Ray; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry; D057166:High-Throughput Screening Assays; D008958:Models, Molecular; D008968:Molecular Conformation; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism; D019809:Streptavidin / Q000737:chemistry; D000637:Transaminases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D001710:Biotin; C004749:desthiobiotin; D019809:Streptavidin; D000637:Transaminases; C102100:BioA protein, bacteria