The covalent attachment of biotin onto certain metabolic enzymes is a post-translational modification that occurs throughout the living world. Biotin protein ligase (BPL) is the enzyme responsible for this process. Crystal structures of class I and II BPLs, present in archaea and bacteria, have been reported. However, the class III BPLs, found in mammals, fungi and insects, have not been extensively characterised. Class III BPLs contain a catalytic domain that is conserved between the 3 classes, but also contain a large N-terminal extension that assists with selection of appropriate biotinylation targets. The paucity of structural information means the molecular basis of the N-terminal domain in substrate recognition is unknown. Crystallography attempts have so far been unsuccessful. Therefore, we have applied alternative techniques to gain new insights into the structure and function of the class III Saccharomyces cerevisiae BPL (ScBPL). Homology modelling using Phyre suggests class III N‑terminal domains share the structural fold of the glutamine amidotransferase (GnAT) subunit from the pyridoxal-5ʹ-phosphate synthase complex. GnATs catalyse the conversion of glutamine to glutamate, and the catalytic triad of residues responsible are conserved and correctly positioned in the ScBPL N-terminal domain model. Preliminary 1D-NMR demonstrated ScBPL does not possess this catalytic activity, and enzyme assays suggest glutamine has no effect on ScBPL activity. Ligand observed NMR assays are being conducted to establish whether ScBPL is capable of binding glutamine, without enzymatic conversion. Mass spectrometry surface labelling and crosslinking techniques, together with SAXS, are being used to validate homology models and provide structural insights into these class III BPLs. These strategies, together with ion-mobility MS and NMR spectroscopy, are also being employed to generate a structural view of how the N‑terminal domain interacts with substrate. Understanding these interactions will provide valuable insights into this precise post-translational modification.