Abstract
Exopeptidases, including dipeptidyl- and tripeptidylpeptidase, are crucial for the growth of Porphyromonas gingivalis, a periodontopathic asaccharolytic bacterium that incorporates amino acids mainly as di- and tripeptides. In this study, we identified a novel exopeptidase, designated acylpeptidyl oligopeptidase (AOP), composed of 759 amino acid residues with active Ser(615) and encoded by PGN_1349 in P. gingivalis ATCC 33277. AOP is currently listed as an unassigned S9 family peptidase or prolyl oligopeptidase. Recombinant AOP did not hydrolyze a Pro-Xaa bond. In addition, although sequence similarities to human and archaea-type acylaminoacyl peptidase sequences were observed, its enzymatic properties were apparently distinct from those, because AOP scarcely released an N-acyl-amino acid as compared with di- and tripeptides, especially with N-terminal modification. The kcat/Km value against benzyloxycarbonyl-Val-Lys-Met-4-methycoumaryl-7-amide, the most potent substrate, was 123.3 ± 17.3 μm(-1) s(-1), optimal pH was 7-8.5, and the activity was decreased with increased NaCl concentrations. AOP existed predominantly in the periplasmic fraction as a monomer, whereas equilibrium between monomers and oligomers was observed with a recombinant molecule, suggesting a tendency of oligomerization mediated by the N-terminal region (Met(16)-Glu(101)). Three-dimensional modeling revealed the three domain structures (residues Met(16)-Ala(126), which has no similar homologue with known structure; residues Leu(127)-Met(495) (β-propeller domain); and residues Ala(496)-Phe(736) (α/β-hydrolase domain)) and further indicated the hydrophobic S1 site of AOP in accord with its hydrophobic P1 preference. AOP orthologues are widely distributed in bacteria, archaea, and eukaryotes, suggesting its importance for processing of nutritional and/or bioactive oligopeptides.
Highlights
Porphyromonas gingivalis, a Gram-negative obligate anaerobe, has been implicated as the causative agent of chronic periodontal disease (1, 2), which is a major reason for permanent tooth loss (3–5)
PGN_1349 Responsible for Remaining Activity in NDP212— We previously reported that Met-Leu-MCA-hydrolyzing activity was markedly decreased in a dpp4-5-7 triple knock-out P. gingivalis strain (NDP211)
These results indicated that the bacterium expresses a peptidase related to MetLeu-MCA hydrolysis in addition to DPP5 and DPP7 and suggested an up-regulation mechanism to compensate for the loss of dipeptide production
Summary
Arg-gingipain; Kgp, Lys-gingipain; DPP, dipeptidyl-peptidase; PTP-A, prolyl tripeptidylpeptidase A; AOP, acylpeptidyl oligopeptidase; AAP, acylaminoacyl peptidase; MCA, 4-methycoumaryl-7-amide; Z-, benzyloxycarbonyl-; Suc-, succinyl-; Boc-, t-butyloxycarbonyl- ((2S)-2-amino-3-(benzyloxycarbonyl) propionyl-); TLCK, N␣-p-tosylL-lysine chloromethyl ketone; DPF, dipeptidylpeptidase family member. Product (plasmid or DNA fragment) pTrcHis2TOPO-PGN1349 pTrcHis2TOPO-PGN1349 pQE60-PGN1542 pQE60-PGN1542 pQE60-PGN1878 pQE60-PGN1878 pTrcHis2TOPO-PGN1349-N102 pTrcHis2TOPO-PGN1349-N102 pTrcHis2TOPO-PGN1349-S615A pTrcHis2TOPO-PGN1349-N102-S615A PGN1349 5Ј region with cepA anchor PGN1349 5Ј region with cepA anchor cepA with 5PGN1349 anchor cepA with 5PGN1349 anchor 5PGN1349 3Ј region with cepA anchor 5PGN1349 3Ј region with cepA anchor 5PGN1349-cepA 5PGN1349-cepA ence for the P1 (15, 25) as well as P2 (26) positions, whereas we discovered that DPP11 (S46.002) is a unique DPP specific for acidic P1 residues (Asp and Glu) (27) In addition to these DPPs, P. gingivalis possesses a metallopeptidase encoded by the gene PGN_1645, which was identified as DPP3 (M49.001) and specific for P1 Arg. DPP3 appears to be localized in the cytosol, whereas the Arg-specific DPP activity of Rgp plays a role in extracellular substrate processing (24). The observed peptidase properties of PGN_1349 indicated it as a novel oligopeptidase, designated acylpeptidyl oligopeptidase (AOP), with a preference for hydrophobic amino acids at the P1 position of its substrates, especially those with N-terminal modification
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
