Phenylalanine 664 of dipeptidyl peptidase (DPP) 7 and Phenylalanine 671 of DPP11 mediate preference for P2‐position hydrophobic residues of a substrate

Abstract

Dipeptidyl peptidases (DPPs) are crucial for the energy metabolism in Porphyromonas gingivalis, a Gram-negative proteolytic and asaccharolytic anaerobic rod causing chronic periodontitis. Three DPPs, DPPIV specific for Pro, DPP7 for hydrophobic residues and DPP11 for Asp/Glu at the P1 position, are expressed in the bacterium. Like DPP7, DPP11 belongs to the S46 protease family, and they share 38.7% sequence identity. Although DPP11 is preferential for hydrophobic residues at the P2 position, it has been reported that DPP7 has no preference at the P2 position. In the present study, we defined the detailed P2 substrate preference of DPP7 and the amino acid residue responsible for the specificity. DPP7 most efficiently hydrolyzed Met-Leu-dipeptidyl-4-methylcoumaryl-7-amide (MCA) carrying hydrophobic residues at the P1 position with kcat/Km of 10.62 ± 2.51 μM−1 s−1, while it unexpectedly cleaved substrates with hydrophilic (Gln, Asn) or charged (Asp, Arg) residues. Examination with 21 dipeptidyl MCA demonstrated that DPP7-peptidase activity was dependent on hydrophobicity of the P2- as well as P1-position residue, thus it correlated best with the sum of the hydrophobicity index of P1- and P2-amino acid residues. Hydrophobicity of the P1 and P2 positions ensured efficient enzyme catalysis by increasing kcat and lowering Km values, respectively. Substitution of hydrophobic residues conserved in the S46 DPP7/DPP11 family to Ala revealed that Phe664 of DPP7 and Phe671 of DPP11 primarily afforded hydrophobic P2 preference. A modeling study suggested that Phe664 and Gly666 of DPP7 and Phe671 and Arg673 of DPP11 being associated with the P2- and P1-position residues, respectively, are located adjacent to the catalytic Ser648/Ser655. The present results expand the substrate repertoire of DPP7, which ensures efficient degradation of oligopeptides in asaccharolytic bacteria.

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

“…Subsequently, oligopeptides are converted in periplasmic space into di-and tripeptides by DPPs and PTP-A, respectively, with DPP5 and DPP7 functioning with oligopeptides with hydrophobic residues at the second position from the N terminus. Additionally, the P2 position hydrophobic residue enhances the activities of DPP7 (26). Those with Pro at the second and third positions are cleaved by DPP4 and PTP-A, respectively.…”

“…These are serine peptidases belonging to either the S9 or S46 family, with DPP4 (S09.003) showing a preference for Pro at P1 (13,14) and DPP5 (S09.012), the first entity identified in bacteria, showing a preference for hydrophobic P1 residues and no specificity at the P2 position (24). Furthermore, DPP7 (S46.001) has a hydrophobic prefer-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.…”

“…AOP at 78 kDa was predominantly detected in the periplasm/outer membrane and cytosol fractions. When the periplasmic/outer membrane fraction was subjected to size exclusion HPLC, 78-kDa AOP was detected as a monomer at fractions [23][24][25][26]. Because the cytosolic preparation contained a considerable amount of periplasmic components, P. gingivalis AOP appears to be primarily distributed in the periplasm and present mainly as a monomer in cells.…”

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

“…This profile could be explained by actions of the 3 defined DPPs: i.e. Gly-Pro-MCA by DPP4; LysAla-MCA by both DPP7 and DPP4; Met-Leu-, Gly-Phe-, LeuArg-, and Ser-Tyr-MCA by DPP7; and Leu-Asp-, Leu-Glu-, and Val-Asp-MCA by DPP11, whereas Leu-Gln-MCA may be cleaved by DPP7 because of its hydrophobic P2 preference (33). Several substrates such as Thr-Ser-and Gly-Gly-MCA were scarcely released by the bacterium, as suspected based on the substrate preferences of the 3 DPPs and a previous report that noted that no peptidase was found to be responsible for Thr, Ser, and Gly in P. gingivalis (34).…”

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

“…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). These peptidases are thought to cover most combinations of P2 and P1 amino acid residues and, therefore, efficiently produce N-terminal dipeptides from polypeptides (21).…”