Split β-barrel proteins are mostly uncharacterised, though they are abundant in pathogenic Mycobacteria including multidrug-resistant strains of Mycobacterium tuberculosis.  The cofactor preferences of these proteins for FMN, FAD, F₄₂₀ and heme influence their diverse functional roles as oxidases, reductases and oxygenases despite their structural similarity. However, only a few have known functions, and any contribution of most members of this family to mycobacterial virulence or the reason for their abundance remains unclear. For preliminary functional annotation, mycobacterial FMN, FAD, F₄₂₀ and heme binding split β-barrel proteins have been experimentally distinguished and 4 novel structures were solved, including the first identified mycobacterial FAD binding proteins from this family. This has been used as a basis for ancestral protein resurrection, used to understand how these proteins have evolved to utilise the rare cofactor F₄₂₀ instead of the ubiquitous flavin cofactors FMN and FAD. The classification of these proteins and analysis of their genomic-context has led to the discovery of F₄₂₀-dependent fatty acid reductases within this family, as it forms the basis for identifying their physiological roles and contribution to mycobacterial virulence.