Evolution of the Cytochrome P450 Family 4 (CYP4) Subfamilies in Birds

Cytochrome P450 (CYP) genes form a large and functionally diverse superfamily of heme-thiolate monooxygenases that catalyse the oxidation of endogenous and exogenous compounds. Within this superfamily, CYP4 enzymes primarily act on fatty acids, substrates central to energy metabolism. Sustained flight and long-distance migration in birds rely on efficient use of fatty acids, making CYP4 a pertinent system for examining how lipid-associated functions may vary across avian lineages. Across Aves, species occupy diverse ecological niches and display substantial variation in metabolic demands. Here, we characterised CYP4 evolution across birds using 363 avian whole genomes, which cover over 90% of bird families, to investigate how gene variation relates to differences in ecological traits and metabolic strategies. Using this extensive genomic dataset, we identified 4 CYP4 subfamilies and analysed their patterns of sequence evolution. The analyses revealed conserved elements of the avian CYP4 repertoire alongside lineage-specific variation. Several amino-acid positions under positive selection were located within substrate recognition sites (SRS), regions that influence substrate binding and catalytic properties. Changes at these positions may reflect shifts in enzymatic function associated with differences in ecological or physiological traits among species. Overall, the results highlight SRS-associated variation within CYP4 that may reflect adaptive responses to environmental change. These findings advance understanding of how lipid-associated metabolic pathways have been shaped during avian diversification and provide insight into the evolutionary history of a CYP family linked to metabolic adaptation in birds.