Discrimination based on Gly and Arg/Ser at position 673 between dipeptidyl-peptidase (DPP) 7 and DPP11, widely distributed DPPs in pathogenic and environmental gram-negative bacteria

Abstract

Porphyromonas gingivalis, an asaccharolytic gram-negative rod-shaped bacterium, expresses the novel Asp/Glu-specific dipeptidyl-peptidase (DPP) 11 (Ohara-Nemoto, Y. et al., (2011) J. Biol. Chem. 286, 38115-38127), which has been categorized as a member of the S46/DPP7 family that is preferential for hydrophobic residues at the P1 position. From that finding, 129 gene products constituting five clusters from the phylum Bacteroidetes have been newly annotated to either DPP7 or DPP11, whereas the remaining 135 members, mainly from the largest phylum Proteobacteria, have yet to be assigned. In this study, the substrate specificities of the five clusters and an unassigned group were determined with recombinant DPPs from typical species, i.e., P. gingivalis, Capnocytophaga gingivalis, Flavobacterium psychrophilum, Bacteroides fragilis, Bacteroides vulgatus, and Shewanella putrefaciens.Consequently, clusters 1, 3, and 5 were found to be DPP7 with rather broad substrate specificity, and clusters 2 and 4 were DPP11. An unassigned S. putrefaciens DPP carrying Ser 673 exhibited Asp/Glu-specificity more preferable to Glu, in contrast to the Asp preference of DPP11 with Arg 673 from Bacteroidetes species. Mutagenesis experiments revealed that Arg 673 /Ser 673 were indispensable for the Asp/Glu-specificity of DPP11, and that the broad specificity of DPP7 was mediated by Gly 673 . Taken together with the distribution of the two genes, all 264 members of the S46 family could be attributed to either DPP7 or DPP11 by an amino acid at position 673. A more compelling phylogenic tree based on the conserved C-terminal region suggested two gene duplication events in the phylum Bacteroidetes, one causing the development of DPP7 and DPP11 with altered substrate specificities, and the other producing an additional DPP7 in the genus Bacteroides.

Degradation of Incretins and Modulation of Blood Glucose Levels by Periodontopathic Bacterial Dipeptidyl Peptidase 4

“…Together with the present and previous observations of PgDPP4, including the kinetic parameters of enzymatic reactions, optimal pH, inhibitor profiles, and substrate preference for Pro and less for Ala (15,18,31), our results led us to conclude that the enzymatic properties of bacterial DPP4 substantially resemble those of the human entity (28,38). In fact, the present findings revealed release of the N-terminal dipeptide from the incretin peptides GLP-1 and GIP by bacterial DPP4.…”

“…In addition, molecular masses of recombinant and native forms of DPP4 were estimated as approximately 9% smaller than those of the deduced sequences. This difference was not fully explained by the deletion of their signal sequences; however, this difference seems to be an intrinsic property of bacterial DPPs migrating on SDS-PAGE, since reductions of apparent masses on SDS-PAGE have been commonly observed for P. gingivalis DPP5 (15%) (15), DPP7 (10%) (31), and DPP11 (8%) (14).…”

“…The PCR fragment of TfDPP4 was cloned into pTrcHis2 TOPO and that of PiDPP4 digested with BamHI was inserted into the BamHI site of pQE60. The expression plasmid of PgDPP4 Asp 23 to Leu 723 was previously reported (31). All constructs were confirmed by DNA sequencing.…”

“…PgDPP4 potently hydrolyzes Gly-Pro-4-methylcoumaryl-7-amide (MCA) and Lys-Ala-MCA as well at a much lower level of activity (31), the same manner as mammalian DPP4, and that activity is efficiently suppressed by hDPP4 inhibitors (15). These similarities between PgDPP4 and hDPP4 led us to speculate that PgDPP4 mimics the role of hDPP4 in glycemic control during recurrent bacteremia in periodontal patients, which may explain the link between severe periodontitis and diabetes mellitus.…”

Identification and Characterization of Prokaryotic Dipeptidyl-peptidase 5 from Porphyromonas gingivalis

“…DPP4, DPP7, and DPP11, have been identified, of which DPP4 is highly specific for Pro and less potently for Ala at the penultimate position from the N terminus (P1) (MEROPS classification: S9.013) (21), whereas DPP7 prefers aliphatic and aromatic residues (22). We recently reported that DPP7 also releases dipeptides with a non-hydrophobic P1 residue, if the N-terminal (P2) residue is hydrophobic (23). The third and novel enzyme DPP11 (S46.002) shares a 38.7% amino acid sequence identity with DPP7 (S46.001), and is specific for Asp and Glu (24).…”