title: Escherichia coli biotin protein ligase: characterization and development of a high-throughput assay.
abstract: The 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.
authors.affiliation: School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia 5005, Australia.
authors.forename: Belinda
authors.initials: B
authors.lastname: Ng
authors.forename: Steven W
authors.initials: SW
authors.lastname: Polyak
authors.forename: Daniel
authors.initials: D
authors.lastname: Bird
authors.forename: Lisa
authors.initials: L
authors.lastname: Bailey
authors.forename: John C
authors.initials: JC
authors.lastname: Wallace
authors.forename: Grant W
authors.initials: GW
authors.lastname: Booker
mesh_terms: D019731:Carbon-Nitrogen Ligases / Q000378:metabolism*; D002623:Chemistry Techniques, Analytical / Q000379:methods; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007700:Kinetics; D012097:Repressor Proteins / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli