Structural and kinetic characterisation of class III biotin protein ligases; novel anti fungal drug targets

Sternicki LM1, Nguyen S1, Pukala TL2, Pendini NR1,3, Beckham S3, Wilce MC3, Booker GW1, Wegener KL1 and Polyak SW1

  1. School of Biological Sciences, The University of Adelaide, North Terrace, Adelaide, South Australia, Australia 5005.
  2. School of Physical Sciences, The University of Adelaide, North Terrace, Adelaide, South Australia, Australia 5005.
  3. School of Biological Sciences, Monash University, Clayton, Victoria, Australia 3800.

The covalent post-translational attachment of biotin is necessary for the activity of certain metabolic enzymes. Biotin protein ligase (BPL) is responsible for this modification and has been proposed as a novel anti-infective target. Crystal structures of class I and II BPLs, present in archaea and bacteria, have been reported and have aided the design of inhibitors against bacterial BPLs. However, the class III BPLs, found in mammals, fungi and insects, have not been extensively characterised nor exploited for antifungal therapeutics. These BPLs differ as they contain a large N-terminal extension that is proposed to assist selection of appropriate biotinylation targets. Limited structural information, including the absence of a class III BPL crystal structure, has hindered the molecular understanding of substrate recognition by the N-terminal extension and the development of antifungal inhibitors. Initial kinetic characterisation of four class III BPLs, namely those from the fungi Saccharomyces cerevisiae, Candida albicans, Botrytis cinerea and Zymoseptoria tritici, revealed different KM values for the substrates. Likewise, inhibition constants for known substrate mimics varied between the enzymes. These data reveal subtle structural differences localised around the substrate binding sites between these class III enzymes, suggesting selective inhibition may be possible. The S. cerevisiae BPL was more thoroughly investigated for structural insights, as crystallography of these four BPLs has so far been unsuccessful. SAXS, ion mobility MS and hydrogen-deuterium exchange MS are being employed to investigate conformational changes and enzyme dynamics associated with ligand binding. Crosslinking MS is also being utilised to delineate how the N-terminal domain facilitates interactions with substrates targeted for biotinylation. This information will aid the refinement of homology models of S. cerevisiae BPL to provide a model of a class III BPL structure. This structural information is vital for the development of selective anti-fungal inhibitors that target pathogenic BPLs but not the human isoform.