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. Class III BPLs contain an additional N-terminal extension that is proposed to assist selection of appropriate biotinylation targets. Limited structural information, including the absence of a crystal structure, has hindered the molecular understanding of substrate recognition by the N-terminal extension and the development of antifungal inhibitors. Whilst crystallography trials of the fungal class III BPLs are ongoing, mass spectrometry techniques have been utilised to gain structural insight into this enzyme and its conformational dynamics upon ligand binding. Ion mobility MS and collision induced unfolding MS revealed no gross conformational changes to the structure or stability when ligands are bound. However, the higher resolution technique of hydrogen-deuterium exchange MS revealed conformational changes in the ligand binding site and allosteric rearrangements in the unique N-terminal extension. Crosslinking MS is now being utilised to delineate which regions of the N-terminal extension are involved in facilitating interactions with the substrates targeted for biotinylation, and whether these interactions are responsible for the substrate preferences demonstrated by activity assays. These data will allow the refinement of homology models of the Saccharomyces cerevisiae BPL to provide a model of a class III BPL structure in complex with substrate. Ultimately, this structural information is vital for the development of selective anti-fungal inhibitors that target pathogenic BPLs over the human isoform.