Reverse Conservation Analysis Reveals the Specificity Determining Residues of Cytochrome P450 Family 2 (CYP 2)

The concept of conservation of amino acids is widely used to identify important alignment positions of orthologs. The assumption is that important amino acid residues will be conserved in the protein family during the evolutionary process. For paralog alignment, on the other hand, the opposite concept can be used to identify residues that are responsible for specificity. Assuming that the function-specific or ligand-specific residue positions will have higher diversity since they are under evolutionary pressure to fit the target specificity, these function-specific or ligand-specific residues positions will have a lower degree of conservation than other positions in a highly conserved paralog alignment. This study assessed the ability of reverse conservation analysis to identify function-specific and ligand-specific residue positions in closely related paralog. Reverse conservation analysis of paralog alignments successfully identified all six previously reported substrate recognition sites (SRSs) in cytochrome P450 family 2 (CYP 2). Further analysis of each subfamily identified the specificity-determining residues (SDRs) that have been experimentally found. New potential SDRs were also predicted and await confirmation by further experiments or modeling calculations. This concept may be also applied to identify SDRs in other protein families.