A Porphyromonas gingivalis Periplasmic Novel Exopeptidase, Acylpeptidyl Oligopeptidase, Releases N-Acylated Di- and Tripeptides from Oligopeptides

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-acylamino acid as compared with di-and tripeptides, especially with N-terminal modification. The k cat /K m value against benzyloxycarbonyl-Val-Lys-Met-4-methycoumaryl-7-amide, the most potent substrate, was 123.3 ؎ 17.3 M ؊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 (␤-propeller domain); and residues Ala 496 -Phe 736 (␣/␤-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.

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

“…Since GLP-1 (M r , 3,355.7) and GIP (M r , 4,983.6) are likely metabolized in the periplasmic space, molecules with a molecular mass smaller than 5,000 permeate through the outer membrane. Similarly, this and previous examinations have demonstrated interactions among synthetic oligopeptidyl MCA substrates (M r , 320 ϳ 764), including hydrophobic, anionic, and cationic amino acid residues at the P1 and P2 positions, and P. gingivalis periplasm-localized exopeptidases (14)(15)(16).…”

“…Bacterial DPP4 activity was cell associated, while no activity was detected in the culture supernatants of the three microorganisms. Our previous studies of DPP5 (15), DPP11 (14), and AOP (16) indicated that these exopeptidases are localized as soluble forms in the periplasmic space. Hence, PgDPP4 seems to be also located in the periplasmic space.…”

“…We recently identified novel dipeptide-producing exopeptidases in P. gingivalis, including DPP11, which specifically releases Xaa-Asp and Xaa-Glu (14); DPP5, with preference for the penultimate Ala and hydrophobic residues from the N terminus (15); and acylpeptidyl oligopeptidase (AOP), with preference mainly for the P1 position hydrophobic residue (16). Moreover, P. gingivalis expresses two additional DPPs and gingipains: DPP4 releases mainly Xaa-Pro but also Xaa-Ala and His-Ser (17,18), DPP7 preferentially cleaves dipeptides with both P1 and P2 hydrophobic residues (19,20), and Lys and Arg gingipains (Kgp and Rgp, respectively) exhibit Lysand Arg-specific dipeptidyl peptidase activities as well as endopeptidase activities (15).…”

Identification of a new subtype of dipeptidyl peptidase 11 and a third group of the S46-family members specifically present in the genus Bacteroides

“…(generally referred to as DPPIII) specific for Arg (M49.003) is also present, this DPP does not seem to be involved in extracellular protein metabolism, because DPP3 appears to be located in the cytoplasm [11]. Additionally, acylpeptidyl oligopeptidase (AOP) produces di-and tri-peptides preferentially from N-terminally acylated peptides [16]. Therefore, in consideration of the fact that most extracellular polypeptides derived from human serum are N-terminally acylated, proteinaceous nutrients in gingival crevicular fluid should be effectively degraded into di-and tri-peptides, and then incorporated into P. gingivalis by co-operative actions of these peptidases [17].…”

Dynamic oligopeptide acquisition by the RagAB transporter fromPorphyromonas gingivalis

“…Unlike many human gut Bacteroidetes that specialise in degrading complex host and dietary glycans, P. gingivalis is asaccharolytic and exclusively utilises peptides for growth 8 . Those peptides are generated by multiple proteases, including several secreted endopeptidases and periplasmic di- and tri-peptidyl-peptidases 9,10 . The best-known P. gingivalis endopeptidases are the gingipains, large and abundant surface-anchored cysteine proteases with cumulative trypsin-like activity, which are essential for P. gingivalis virulence and growth on proteins as the sole source of carbon 11 .…”