Biotin protein ligase from Candida albicans: expression, purification and development of a novel assay.
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.
Pendini
Nicole R
NR
School of Molecular and Biomedical Science, University of Adelaide, Molecular Lifesciences Building, North Tce, Adelaide, SA 5005, Australia.
Bailey
Lisa M
LM
Booker
Grant W
GW
Wilce
Matthew C J
MC
Wallace
John C
JC
Polyak
Steven W
SW
D000103:Acetyl-CoA Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000935:Antifungal Agents / Q000737:chemistry; D001681:Biological Assay / Q000379:methods*; D001710:Biotin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics; D019731:Carbon-Nitrogen Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000737:chemistry; D005227:Fatty Acids / Q000096:biosynthesis; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005943:Gluconeogenesis / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D011766:Pyruvate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000935:Antifungal Agents; D004791:Enzyme Inhibitors; D005227:Fatty Acids; D005656:Fungal Proteins; D011994:Recombinant Proteins; D001710:Biotin; D019731:Carbon-Nitrogen Ligases; D011766:Pyruvate Carboxylase; D000103:Acetyl-CoA Carboxylase