Evolutionary divergence of dipeptidyl-peptidase (DPP)11 from ancestral bacterial DPP7

To date, the S46 peptidase family is limited to two members, dipeptidyl-peptidase (DPP)7 and DPP11, which are widely distributed among Gram-negative bacteria and present in both oral and intestinal microbiota. Both peptidases are indispensable for the growth of the periodontopathic bacterium Porphyromonas gingivalis. They have a 40.0% amino acid identity, and exhibit specificity for hydrophobic and acidic P1 residues, respectively. This specificity is associated with S1 Gly⁶⁶⁶ in DPP7 and the equivalent residue, Arg⁶⁷³, in DPP11. Recent studies have revealed a relaxed P1 specificity of DPP7 even for neutral amino acids and hydrophilic Asn. Thus, the broad utility of DPP7 suggests that the ancestral enzyme of the S46 family resembled DPP7. The aim of the present study was to reconstruct the evolutionary divergence of these two DPPs. DPP7 hydrolyzed the tetrapeptidyl substrate LE-|-MP- and LD-|-MP-4-methylcoumaryl-7-amide (MCA) at 4.0% and 7.3%, respectively, of the efficiency of DPP11, whereas DPP11 did not hydrolyze the DPP7 substrate LN-|-MP-MCA, indicating that DPP7 partially complements the role of DPP11. A single amino acid substitution at the S1 site in DPP7 (Gly666Arg) enhanced hydrolysis toward Asp and Glu to a level higher than that of DPP7 wild-type, while a reverse substitution in DPP11 (Arg673Gly) abolished hydrolytic activities toward all substrates examined. Taken together, DPP7 might represent the ancestral form of S46-family peptidases, from which the dpp11 gene arose through gene duplication and subsequent mutations initiated by Gly666Arg substitution.