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

doi:10.1016/j.ab.2011.05.003

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

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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 language	English (US)
Pages (from-to)	27-38
Number of pages	12
Journal	Analytical Biochemistry
Volume	416
Issue number	1
DOIs	https://doi.org/10.1016/j.ab.2011.05.003
State	Published - 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

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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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.",

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

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.). ",

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AU - Manjunatha, Ujjini

AU - Sham, Yuk Y.

AU - Thomas, David D.

AU - Aldrich, Courtney C.

N1 - 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.).

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AB - 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.

KW - Biotin

KW - Dethiobiotin

KW - Fluorescence displacement assay

KW - High-throughput screening

KW - Mycobacterium tuberculosis

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

Abstract

Bibliographical note

Keywords

UN SDGs

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