Cytochrome P450s from the CYP52 family participate in the assimilation of alkanes and fatty acids in fungi. In this work, the evolutionary history of a set of orthologous and paralogous CYP52 proteins from Saccharomycetales yeasts was inferred. Further, the phenotypic assimilation profiles were related with the distribution of cytochrome CYP52 members among species. The maximum likelihood phylogeny of CYP52 inferred proteins reveled a frequent ancient and modern duplication and loss events that generated orthologous and paralogous groups. Phylogeny and assimilation profiles of alkanes and fatty acids showed a family expansion in yeast isolated from hydrophobic-rich environments. Docking analysis of deduced ancient CYP52 proteins suggests that the most ancient function was the oxidation of C4-C11 alkanes, while the oxidation of >10 carbon alkanes and fatty acids is a derived character. The ancient CYP52 paralogs displayed partial specialization and promiscuous interaction with hydrophobic substrates. Additionally, functional optimization was not evident. Changes in the interaction of ancient CYP52 with different alkanes and fatty acids could be associated with modifications in spatial orientations of the amino acid residues that comprise the active site. The extended family of CYP52 proteins is likely evolving toward functional specialization, and certain redundancy for substrates is being maintained. Cytochromes P450 (CYPs) are an extended heme-thiolate enzyme superfamily that are widely distributed among different biological domains 1,2. P450s are involved in the oxidation of myriad endogenous and xenobiotic hydrophobic compounds. Hence, P450s play a critical role in the biosynthesis of structural molecules and secondary metabolites 3,4 , utilization of compounds as sole carbon and energy sources 5 , and cellular detoxification 6 , among others. The cytochrome P450s that belong to the CYP52 family are present in the orders Eurotiales, Pezizomycetes, Leotiomycetes, Dothideomycetes and Saccharomycetales of Ascomycota fungi 7. CYP52 enzymes are located in the endoplasmic reticulum (ER) membrane, and their main function is the hydroxylation of n-alkanes and fatty acids, which are successively oxidized to mono-or dicarboxylic fatty acids, respectively, by additional oxidation reactions catalyzed by alcohol and aldehyde deshydrogenases 5,8. Finally, fatty acids are degraded in the fungal peroxisome and mitochondria via the β-oxidation pathway to CO 2. This degradation produces acetyl-CoA, FADH 2 , and NADH 9-12. Candida albicans, Candida maltosa, Candida tropicalis, and Yarrowia lipolytica are model Saccharomycetales used for the study of function and transcriptional regulation of multiple orthologous and paralogous P450 encoded by CYP52 genes 7,13-18. The CYP52 genes in C. maltosa, Candida pseudoglaebosa, C. tropicalis, Kodamaea
