Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/45307
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dc.contributor.authorNg, B.en
dc.contributor.authorPolyak, S.en
dc.contributor.authorBird, D.en
dc.contributor.authorBailey, L.en
dc.contributor.authorWallace, J.en
dc.contributor.authorBooker, G.en
dc.date.issued2008en
dc.identifier.citationAnalytical Biochemistry, 2008; 376(1):131-136en
dc.identifier.issn0003-2697en
dc.identifier.issn1096-0309en
dc.identifier.urihttp://hdl.handle.net/2440/45307-
dc.description.abstractThe rapid rise in pathogenic bacteria resistant to current treatments, coupled with the paucity of new therapeutic agents in the pipeline, has resulted in a significant need for new antibiotics. One strategy to overcome resistance requires new chemical entities that inhibit key enzymes in essential metabolic processes that have not been previously targeted and for which there is no preexisting drug resistance. Biotin protein ligase (BPL), required to complete acetyl CoA carboxylase's capability for fatty acid biosynthesis, is one target that has not yet been fully explored. However, its application in large-scale compound screens has been limited due to the lack of a truly high-throughput assay for enzyme activity. Here we report a novel assay system for BPL from Escherichia coli (BirA). This assay employs fluorescence polarization technology together with a unique peptide substrate for BirA. Additionally, the multiple handling steps and requirement for radiolabeled ligands associated with previous assays have been eliminated. Kinetic analysis of MgATP (K(m) 0.25+/-0.01 mM) and biotin (K(m) 1.45+/-0.15 microM) binding produced results consistent with published data. Inhibition studies with end products of the BPL reaction, AMP and pyrophosphate, further validated the assay. Statistical analysis, performed upon both intraassay and interassay results (n = 30), showed the coefficient of variance to be <10% across all data sets. Furthermore, Z' factors between 0.5 and 0.8 demonstrated the utility of this technology in high-throughput applications.en
dc.description.statementofresponsibilityBelinda Ng, Steven W. Polyak, Daniel Bird, Lisa Bailey, John C. Wallace and Grant W. Bookeren
dc.language.isoenen
dc.publisherAcademic Press Inc Elsevier Scienceen
dc.rights© 2008 Elsevier Inc. All rights reserved.en
dc.subjectCarbon-Nitrogen Ligases; Escherichia coli Proteins; Repressor Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Kinetics; Chemistry Techniques, Analyticalen
dc.titleEscherichia coli biotin protein ligase: characterization and development of a high-throughput assayen
dc.typeJournal articleen
dc.identifier.rmid0020080526en
dc.identifier.doi10.1016/j.ab.2008.01.026en
dc.identifier.pubid43525-
pubs.library.collectionMolecular and Biomedical Science publicationsen
pubs.verification-statusVerifieden
pubs.publication-statusPublisheden
dc.identifier.orcidPolyak, S. [0000-0002-8458-5194]en
dc.identifier.orcidBooker, G. [0000-0001-7207-4699]en
Appears in Collections:Molecular and Biomedical Science publications

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