Importance of N-terminal P2 residue in substrate recognition by bacterial DPP7 activity revealed by coefficient of...

Dipeptidyl-peptidases (DPPs), including the S9-family DPP4 and DPP5 and the S46-family DPP7 and DPP11, are essential for growth of Porphyromonas gingivalis, a periodontopathic bacterium associated with systemic diseases. DPPs release dipeptides in diverse combinations from peptidyl N-termini, thus enabling utilization of amino acids as carbon and energy sources. This study examined how DPP5 and DPP7 achieve division of labor despite both possessing preference for hydrophobic residues at the P1 and P2 substrate positions. To identify important positions of substrates that define the specificity of DPPs, the terms coefficient of variation in amino acid frequency and coefficient of variation in protease activity (CVA) are introduced, as the values increase when a substrate position more strongly affects enzyme activity. In particular, CVA was found useful for peptidases, such as DPPs, for which a limited number of substrates are available. Using HX-|-LD-4-methylcoumaryl-7-amide (MCA), XN-|-LD-MCA, and XN-|-SD-MCA (X = nine amino acids) as substrates, DPP5 and DPP7 exhibited higher CVA values at the P1 and P2 positions, respectively, than at their counter positions. Furthermore, least-squares method applied on the relationship between peptidase activity and hydrophobicity for 27 dipeptidyl-MCA substrates independently indicated the best fit for DPP5 at 100% P1 and 0% P2, and for DPP7 at 53% P1 and 47% P2. Both approaches showed that functional division of labor is achieved through the exclusive P1 preference of DPP5 and the unusual P2 significance for DPP7. These results also demonstrate the usefulness of CVA for evaluating positional effects of substrates in peptidase reactions.