Abstract
Porphyromonas gingivalis, a Gram-negative asaccharolytic anaerobe, is a major causative organism of chronic periodontitis. Because the bacterium utilizes amino acids as energy and carbon sources and incorporates them mainly as dipeptides, a wide variety of dipeptide production processes mediated by dipeptidyl-peptidases (DPPs) should be beneficial for the organism. In the present study, we identified the fourth P. gingivalis enzyme, DPP5. In a dpp4-7-11-disrupted P. gingivalis ATCC 33277, a DPP7-like activity still remained. PGN_0756 possessed an activity indistinguishable from that of the mutant, and was identified as a bacterial orthologue of fungal DPP5, because of its substrate specificity and 28.5% amino acid sequence identity with an Aspergillus fumigatus entity. P. gingivalis DPP5 was composed of 684 amino acids with a molecular mass of 77,453, and existed as a dimer while migrating at 66 kDa on SDS-PAGE. It preferred Ala and hydrophobic residues, had no activity toward Pro at the P1 position, and no preference for hydrophobic P2 residues, showed an optimal pH of 6.7 in the presence of NaCl, demonstrated Km and kcat/Km values for Lys-Ala-MCA of 688 μM and 11.02 μM(-1) s(-1), respectively, and was localized in the periplasm. DPP5 elaborately complemented DPP7 in liberation of dipeptides with hydrophobic P1 residues. Examinations of DPP- and gingipain gene-disrupted mutants indicated that DPP4, DPP5, DPP7, and DPP11 together with Arg- and Lys-gingipains cooperatively liberate most dipeptides from nutrient oligopeptides. This is the first study to report that DPP5 is expressed not only in eukaryotes, but also widely distributed in bacteria and archaea.
Highlights
Dipeptidyl-peptidases (DPPs) are key factors for amino acid metabolism and bacterial growth of asaccharolytic Porphyromonas gingivalis
Activities toward Gly-Pro-MCA by DPP4, Met-Leu-MCA by DPP7, and Leu-Asp-MCA by DPP11 were demonstrated to some extent in the wild-type, and interestingly, these activities were elevated in KDP136 cells, probably to compensate for the loss of gingipain activities
We identified and characterized P. gingivalis DPP5 encoded by the PGN_0756 gene, which has been annotated as prolyl oligopeptidase
Summary
Dipeptidyl-peptidases (DPPs) are key factors for amino acid metabolism and bacterial growth of asaccharolytic Porphyromonas gingivalis. Examinations of DPP- and gingipain gene-disrupted mutants indicated that DPP4, DPP5, DPP7, and DPP11 together with Arg- and Lysgingipains cooperatively liberate most dipeptides from nutrient oligopeptides This is the first study to report that DPP5 is. When Pro is located at the third position from the N terminus, DPPs do not cleave the Xaa2– Pro bond, prolyl tripeptidase A (PTP-A) cleaves the Pro3–Xaa bond [25] These liberated di- and tripeptides are efficiently incorporated into bacterial cells [8], possibly through oligopeptide transporters [13]. Based on enzymatic properties and amino acid sequence similarity, PGN_0756 was identified as DPP5 (EC ϭ 3.4.14.-, S09.012), which was found to be specific for Ala and hydrophobic amino acids at the P1 position To our knowledge, this is the first report of DPP5 distribution in a prokaryotic organism
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