Biotin protein ligase from Candida albicans: expression, purification and development of a novel assay |
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Authors: | Pendini Nicole R Bailey Lisa M Booker Grant W Wilce Matthew C J Wallace John C Polyak Steven W |
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Institution: | a School of Molecular and Biomedical Science, University of Adelaide, Molecular Lifesciences Building, North Tce, Adelaide, SA 5005, Australia b Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Vic., Australia |
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Abstract: | Biotin protein ligase (BPL) is an essential enzyme responsible for the activation of biotin-dependent enzymes through the covalent attachment of biotin. In yeast, disruption of BPL affects important metabolic pathways such as fatty acid biosynthesis and gluconeogenesis. This makes BPL an attractive drug target for new antifungal agents. Here we report the cloning, recombinant expression and purification of BPL from the fungal pathogen Candida albicans. The biotin domains of acetyl CoA carboxylase and pyruvate carboxylase were also cloned and characterised as substrates for BPL. A novel assay was established thereby allowing examination of the enzyme’s properties. These findings will facilitate future structural studies as well as screening efforts to identify potential inhibitors. |
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Keywords: | Biotin protein ligase Candida albicans Fungicide drug discovery Protein structure and function Assay development |
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