Biotin protein ligase from Candida albicans: expression, purification and development of a novel assay

Arch Biochem Biophys. 2008 Nov 15;479(2):163-9. doi: 10.1016/j.abb.2008.08.021. Epub 2008 Sep 11.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetyl-CoA Carboxylase / chemistry
  • Acetyl-CoA Carboxylase / genetics
  • Acetyl-CoA Carboxylase / metabolism
  • Antifungal Agents / chemistry
  • Biological Assay / methods*
  • Biotin / chemistry*
  • Biotin / genetics
  • Biotin / metabolism
  • Candida albicans / enzymology*
  • Candida albicans / genetics
  • Carbon-Nitrogen Ligases / antagonists & inhibitors
  • Carbon-Nitrogen Ligases / chemistry*
  • Carbon-Nitrogen Ligases / metabolism
  • Drug Evaluation, Preclinical / methods
  • Enzyme Inhibitors / chemistry
  • Fatty Acids / biosynthesis
  • Fungal Proteins / chemistry*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Gluconeogenesis / physiology
  • Protein Structure, Tertiary / physiology
  • Pyruvate Carboxylase / chemistry
  • Pyruvate Carboxylase / genetics
  • Pyruvate Carboxylase / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism

Substances

  • Antifungal Agents
  • Enzyme Inhibitors
  • Fatty Acids
  • Fungal Proteins
  • Recombinant Proteins
  • Biotin
  • Carbon-Nitrogen Ligases
  • Pyruvate Carboxylase
  • Acetyl-CoA Carboxylase