Potential elevation of exopeptidase activity of Glu-specific endopeptidase I/GluV8 mediated by hydrophobic P1'-position amino acid residue.

We recently reported that the activities of dipeptidyl-peptidase (DPP)7 and DPP11, S46-family exopeptidases were significantly elevated by the presence of prime-side amino acid residues of substrates caused by an increase in kcat [Ohara-Nemoto Y. et al., J Biol Chem 298(3):101585. doi: 10.1016/j.jbc.2022]. In the present study, the effects of prime-side residues on Glu-specific endopeptidase I/GluV8 from Staphylococcus aureus were investigated using a two-step cleavage method with tetrapeptidyl-methycoumaryl-7-amide (MCA) carrying P2- to P2'-position residues coupled with DPP11 as the second enzyme. GluV8 showed maximal activity toward benzyloxycarbonyl (Z)-LLE-MCA, while the effects of hydrolysis of substrates one residue shorter, such as acetyl (Ac)-Val-Glu- and Leu-Glu-MCA, were negligible. Nevertheless, activity towards Ac-VE-|-ID-MCA, a substrate carrying P1' and P2' residues, emerged and reached a level 44 % of that for Z-LLE-MCA. Among 11 Ac-HAXD-MCA (X is a varied amino acid), the highest level of activity enhancement was achieved with P1'-Leu and Ile, followed by Phe, Val, Ser, Tyr, and Ala, while Gly and Lys showed scant effects. This activation order was in parallel with the hydrophobicity indexes of these amino acids. The prime-side residues increased kcat/KM primarily through a maximum 500-fold elevation of kcat as well as S46-family exopeptidases. The MEROPS substrate database also indicates a close relationship between activity and hydrophobicity of the P1' residues in 93 N-terminal-truncated substrates, though no correlation was observed among all 4328 GluV8 entities examined. Taken together, these results are the first to demonstrate N-terminal exopeptidase activity of GluV8, considered to be prompted by hydrophobic P1' amino acid residues.

Immunoelectron Microscopic Analysis of Dipeptidyl-Peptidases and Dipeptide Transporter Involved in Nutrient Acquisition in Porphyromonas gingivalis.

Porphyromonas gingivalis is an asaccharolytic, Gram-negative, anaerobic bacterium representing a keystone pathogen in chronic periodontitis. The bacterium's energy production depends on the metabolism of amino acids, which are predominantly incorporated as dipeptides via the proton-dependent oligopeptide transporter (Pot). In this study, the localization of dipeptidyl-peptidases (DPPs) and Pot was investigated for the first time in P. gingivalis using immunoelectron microscopy with specific antibodies for the bacterial molecules and gold-conjugated secondary antibodies on ultrathin sections. High-temperature protein G and hemin-binding protein 35 were used as controls, and the cytoplasmic localization of the former and outer membrane localization of the latter were confirmed. P. gingivalis DPP4, DPP5, DPP7, and DPP11, which are considered sufficient for complete dipeptide production, were detected in the periplasmic space. In contrast, DPP3 was localized in the cytoplasmic space in accord with the absence of a signal sequence. The inner membrane localization of Pot was confirmed. Thus, spatial integration of the nutrient acquisition system exists in P. gingivalis, in which where dipeptides are produced in the periplasmic space by DPPs and readily transported across the inner membrane via Pot.

Expanded substrate specificity supported by P1' and P2' residues enables bacterial dipeptidyl-peptidase 7 to degrade bioactive peptides.

Dipeptide production from extracellular proteins is crucial for Porphyromonas gingivalis, a pathogen related to chronic periodontitis, because its energy production is entirely dependent on the metabolism of amino acids predominantly incorporated as dipeptides. These dipeptides are produced by periplasmic dipeptidyl-peptidase (DPP)4, DPP5, DPP7, and DPP11. Although the substrate specificities of these four DPPs cover most amino acids at the penultimate position from the N terminus (P1), no DPP is known to cleave penultimate Gly, Ser, Thr, or His. Here, we report an expanded substrate preference of bacterial DPP7 that covers those residues. MALDI-TOF mass spectrometry analysis demonstrated that DPP7 efficiently degraded incretins and other gastrointestinal peptides, which were successively cleaved at every second residue, including Ala, Gly, Ser, and Gln, as well as authentic hydrophobic residues. Intravenous injection of DPP7 into mice orally administered glucose caused declines in plasma glucagon-like peptide-1 and insulin, accompanied by increased blood glucose levels. A newly developed coupled enzyme reaction system that uses synthetic fluorogenic peptides revealed that the P1' and P2' residues of substrates significantly elevated kcat values, providing an expanded substrate preference. This activity enhancement was most effective toward the substrates with nonfavorable but nonrepulsive P1 residues in DPP7. Enhancement of kcat by prime-side residues was also observed in DPP11 but not DPP4 and DPP5. Based on this expanded substrate specificity, we demonstrate that a combination of DPPs enables proteolytic liberation of all types of N-terminal dipeptides and ensures P. gingivalis growth and pathogenicity.

Dipeptidyl-peptidases: Key enzymes producing entry forms of extracellular proteins in asaccharolytic periodontopathic bacterium Porphyromonas gingivalis.

Porphyromonas gingivalis, a pathogen of chronic periodontitis, is an asaccharolytic microorganism that solely utilizes nutritional amino acids as its energy source and cellular constituents. The bacterium is considered to incorporate proteinaceous nutrients mainly as dipeptides, thus exopeptidases that produce dipeptides from polypeptides are critical for survival and proliferation. We present here an overview of dipeptide production by P. gingivalis mediated by dipeptidyl-peptidases (DPPs), e.g., DPP4, DPP5, DPP7, and DPP11, serine exopeptidases localized in periplasm, which release dipeptides from the N-terminus of polypeptides. Additionally, two other exopeptidases, acylpeptidyl-oligopeptidase (AOP) and prolyl tripeptidyl-peptidase A (PTP-A), which liberate N-terminal acylated di-/tri-peptides and tripeptides with Pro at the third position, respectively, provide polypeptides in an acceptable form for DPPs. Hence, a large fraction of dipeptides is produced from nutritional polypeptides by DPPs with differential specificities in combination with AOP and PTP-A. The resultant dipeptides are then incorporated across the inner membrane mainly via a proton-dependent oligopeptide transporter (POT), a member of the major facilitator superfamily. Recent studies also indicate that DPP4 and DPP7 directly link between periodontal and systemic diseases, such as type 2 diabetes mellitus and coagulation abnormality, respectively. Therefore, these dipeptide-producing and incorporation molecules are considered to be potent targets for prevention and treatment of periodontal and related systemic diseases.

Preferential dipeptide incorporation of Porphyromonas gingivalis mediated by proton-dependent oligopeptide transporter (Pot).

Multiple dipeptidyl-peptidases (DPPs) are present in the periplasmic space of Porphyromonas gingivalis, an asaccharolytic periodontopathic bacterium. Dipeptides produced by DPPs are presumed to be transported into the bacterial cells and metabolized to generate energy and cellular components. The present study aimed to identify a transporter responsible for dipeptide uptake in the bacterium. A real-time metabolic analysis demonstrated that P. gingivalis preferentially incorporated Gly-Xaa dipeptides, and then, single amino acids, tripeptides and longer oligopeptides to lesser extents. Heterologous expression of the P. gingivalis serine/threonine transporter (SstT; PGN_1460), oligopeptide transporter (Opt; PGN_1518) and proton-dependent oligopeptide transporter (Pot; PGN_0135) genes demonstrated that Escherichia coli expressing Pot exclusively incorporated Gly-Gly, while SstT managed Ser uptake and Opt was responsible for Gly-Gly-Gly uptake. Dipeptide uptake was significantly decreased in a P. gingivalis Δpot strain and further suppressed in a Δpot-Δopt double-deficient strain. In addition, the growth of the Δpot strain was markedly attenuated and the Δpot-Δopt strain scarcely grew, whereas the ΔsstT strain grew well almost like wild type. Consequently, these results demonstrate that predominant uptake of dipeptide in P. gingivalis is mostly managed by Pot. We thus propose that Pot is a potential therapeutic target of periodontal disease and P. gingivalis-related systemic diseases.

Characterization of substrate specificity and novel autoprocessing mechanism of dipeptidase A from Prevotella intermedia.

Prevotella intermedia, a Gram-negative anaerobic rod, is frequently observed in subgingival polymicrobial biofilms from adults with chronic periodontitis. Peptidases in periodontopathic bacteria are considered to function as etiological reagents. Prevotella intermedia OMA14 cells abundantly express an unidentified cysteine peptidase specific for Arg-4-methycoumaryl-7-amide (MCA). BAU17746 (locus tag, PIOMA14_I_1238) and BAU18827 (locus tag, PIOMA14_II_0322) emerged as candidates of this peptidase from the substrate specificity and sequence similarity with C69-family Streptococcus gordonii Arg-aminopeptidase. The recombinant form of the former solely exhibited hydrolyzing activity toward Arg-MCA, and BAU17746 possesses a 26.6% amino acid identity with the C69-family Lactobacillus helveticus dipeptidase A. It was found that BAU17746 as well as L. helveticus dipeptidase A was a P1-position Arg-specific dipeptidase A, although the L. helveticus entity, a representative of the C69 family, had been reported to be specific for Leu and Phe. The full-length form of BAU17746 was intramolecularly processed to a mature form carrying the N-terminus of Cys15. In conclusion, the marked Arg-MCA-hydrolyzing activity in Pre. intermedia was mediated by BAU17746 belonging to the C69-family dipeptidase A, in which the mature form carries an essential cysteine at the N-terminus.

Suppressive effects of N-bisphosphonate in osteoblastic cells mitigated by non-N-bisphosphonate but not by sodium-dependent phosphate cotransporter inhibitor.

There are two types of bisphosphonates (BPs), nitrogen-containing (N-BPs) and those free from nitrogen (non-N-BPs). Although N-BPs show greater inhibition of bone resorption than non-N-BPs, their effects are likely accompanied with inflammation, which non-N-BPs mitigate. We examined the competitive effects of zoledronate (ZOL), an N-BP, and etidronate (ETI), a non-N-BP, in osteoblasts. ZOL, but not ETI, markedly reduced alkaline phosphatase activity and cell viability in osteoblastic MC3T3-E1 and Saos2 cells, while that inhibition was relieved by simultaneous administration of ETI, possibly because of competition with ZOL for cellular uptake. However, phosphonoformate, an inhibitor of the phosphonate transporters SLC20A and SLC34A, did not mitigate the reducing effects of ZOL, suggesting that those transporters are not involved in BP uptake in osteoblastic cells. Additionally, ZOL reduced fibroblastic NIH3T3 and C3H10T1/2 cell viability, which was relieved by administration of both ETI and phosphonoformate. Transporter gene expression levels were significantly lower in osteoblasts as compared with fibroblasts, which may account for the distinct effects of phosphonoformate with different cell types. Together, our results suggest existence of a common uptake route of N-BPs and non-N-BPs into osteoblastic cells that is unrelated to the SLC20A and SLC34A families. SIGNIFICANCE OF THE STUDY: N-BP ZOL was shown to suppress differentiation and viability of osteoblasts. ZOL-induced cell viability suppression was also observed in fibroblasts, which was markedly relieved by addition of the non-N-BP ETI. Additionally, mitigation of the effects of ZOL was achieved with phosphonoformate, a sodium-phosphate cotransporter inhibitor, in fibroblastic cells but not osteoblasts. Expression levels of SLC20A and SLC34A family genes were significantly lower in osteoblasts as compared with fibroblasts. These observations suggest that incorporation of N-BPs and non-N-BPs in osteoblasts is mediated via common transporters that appear to be distinct from SLC20A and 34A, which operate in fibroblasts.

Characterization of bacterial acylpeptidyl-oligopeptidase.

Acylpeptidyl-oligopeptidase (AOP), which has been recently identified as a novel enzyme in a periodontopathic bacterium, Porphyromonas gingivalis, removes di- and tri-peptides from N-terminally acylated polypeptides, with a preference for hydrophobic P1-position amino acid residues. To validate enzymatic properties of AOP, characteristics of two bacterial orthologues from Bacteroides dorei (BdAOP), a Gram-negative intestinal rod, and Lysinibacillus sphaericus (LsAOP), a Gram-positive soil rod, were determined. Like P. gingivalis AOP (PgAOP), two orthologues more efficiently hydrolyzed N-terminal acylated peptidyl substrates than non-acylated ones. Optimal pH was shifted from 7.0 to 8.9 for N-acylated to 8.5-9.5 for non-acylated substrates, indicating preference for non-charged hydrophobic N-terminal residues. Hydrophobic P1- and P2-position preferences were common in the three AOPs, although PgAOP preferred Leu and the others preferred Phe at the P1 position. In vitro mutagenesis demonstrated that Phe647 in PgAOP was responsible for the P1 Leu preference. In addition, bacterial AOPs commonly liberated acetyl-Ser1-Tyr2 from α-melanocyte-stimulating hormone. Taken together, although these three bacterial AOPs exhibited some variations in biochemical properties, the present study demonstrated the existence of a group of exopeptidases that preferentially liberates mainly dipeptides from N-terminally acylated polypeptides with a preference for hydrophobic P1 and P2-position residues.

Distribution of dipeptidyl peptidase (DPP) 4, DPP5, DPP7 and DPP11 in human oral microbiota-potent biomarkers indicating presence of periodontopathic bacteria.

Dipeptidyl peptidase (DPP) 4, DPP5, DPP7 and DPP11, expressed in the periplasmic space, are crucial for energy production for Porphyromonas gingivalis, an asaccharolytic bacterium that causes periodontal disease. Bacterial DPP4 seems to be involved in regulation of blood glucose level via degradation of incretins. The present study aimed to identify four dpp orthologs in oral microbiota by database searches, and their enzymatic activities in periodontopathic and cariogenic bacteria, as well as oral specimens were determined. Search in the databases suggested that 43 species of 772 taxa possess dpp4 and other dpp genes. Most species are in the genera Bacteroides, Capnocytophaga, Porphyromonas, Prevotella and Tannerella, indicating a limited distribution of dpp orthologs in anaerobic periodontopathic rods. In accordance with those results, activities of all four DPPs were demonstrated in P. gingivalis, Porphyromonas endodontalis and Tannerella forsythia, while they were negligible in Treponema denticola, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans. Furthermore, DPP activities were also detected in subgingival dental plaque at different intensities among individual specimens, while DPP4 activity presumably derived from human entity was solely predominant in saliva samples. These findings demonstrated that DPP activities in dental plaque serve as potent biomarkers to indicate the presence of periodontopathic bacteria.

Establishment of potent and specific synthetic substrate for dipeptidyl-peptidase 7.

Bacterial dipeptidyl-peptidase (DPP) 7 liberates a dipeptide with a preference for aliphatic and aromatic penultimate residues from the N-terminus. Although synthetic substrates are useful for activity measurements, those currently used are problematic, because they are more efficiently degraded by DPP5. We here aimed to develop a potent and specific substrate and found that the kcat/Km value for Phe-Met-methylcoumaryl-7-amide (MCA) (41.40 ±â€¯0.83 µM-1 s-1) was highest compared to Met-Leu-, Leu-Leu-, and Phe-Leu-MCA (1.06-3.77 µM-1 s-1). Its hydrolyzing activity was abrogated in a Porphyromonas gingivalis dpp7-knockout strain. Conclusively, we propose Phe-Met-MCA as an ideal synthetic substrate for DPP7.

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

Peptidase family S46 consists of two types of dipeptidyl-peptidases (DPPs), DPP7 and DPP11, which liberate dipeptides from the N-termini of polypeptides along with the penultimate hydrophobic and acidic residues, respectively. Their specificities are primarily defined by a single amino acid residue, Gly673 in DPP7 and Arg673 in DPP11 (numbering for Porphyromonas gingivalis DPP11). Bacterial species in the phyla Proteobacteria and Bacteroidetes generally possess one gene for each, while Bacteroides species exceptionally possess three genes, one gene as DPP7 and two genes as DPP11, annotated based on the full-length similarities. In the present study, we aimed to characterize the above-mentioned Bacteroides S46 DPPs. A recombinant protein of the putative DPP11 gene BF9343_2924 from Bacteroides fragilis harboring Gly673 exhibited DPP7 activity by hydrolyzing Leu-Leu-4-methylcoumaryl-7-amide (MCA). Another gene, BF9343_2925, as well as the Bacteroides vulgatus gene (BVU_2252) with Arg673 was confirmed to encode DPP11. These results demonstrated that classification of S46 peptidase is enforceable by the S1 essential residues. Bacteroides DPP11 showed a decreased level of activity towards the substrates, especially with P1-position Glu. Findings of 3D structural modeling indicated three potential amino acid substitutions responsible for the reduction, one of which, Asn650Thr substitution, actually recovered the hydrolyzing activity of Leu-Glu-MCA. On the other hand, the gene currently annotated as DPP7 carrying Gly673 from B. fragilis (BF9343_0130) and Bacteroides ovatus (Bovatus_03382) did not hydrolyze any of the examined substrates. The existence of a phylogenic branch of these putative Bacteroides DPP7 genes classified by the C-terminal conserved region (Ser571-Leu700) strongly suggests that Bacteroides species expresses a DPP with an unknown property. In conclusion, the genus Bacteroides exceptionally expresses three S46-family members; authentic DPP7, a new subtype of DPP11 with substantially reduced specificity for Glu, and a third group of S46 family members.

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

Severe periodontitis is known to aggravate diabetes mellitus, though molecular events related to that link have not been fully elucidated. Porphyromonas gingivalis, a major pathogen of periodontitis, expresses dipeptidyl peptidase 4 (DPP4), which is involved in regulation of blood glucose levels by cleaving incretins in humans. We examined the enzymatic characteristics of DPP4 from P. gingivalis as well as two other periodontopathic bacteria, Tannerella forsythia and Prevotella intermedia, and determined whether it is capable of regulating blood glucose levels. Cell-associated DPP4 activity was found in those microorganisms, which was effectively suppressed by inhibitors of human DPP4, and molecules sized 73 kDa in P. gingivalis, and 71 kDa in T. forsythia and P. intermedia were immunologically detected. The kcat/Km values of recombinant DPP4s ranged from 721 ± 55 to 1,283 ± 23 µM-1s-1 toward Gly-Pro-4-methylcoumaryl-7-amide (MCA), while those were much lower for His-Ala-MCA. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis showed His/Tyr-Ala dipeptide release from the N termini of incretins, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide, respectively, with the action of microbial DPP4. Moreover, intravenous injection of DPP4 into mice decreased plasma active GLP-1 and insulin levels, accompanied by a substantial elevation in blood glucose over the control after oral glucose administration. These results are the first to show that periodontopathic bacterial DPP4 is capable of modulating blood glucose levels the same as mammalian DPP4; thus, the incidence of periodontopathic bacteremia may exacerbate diabetes mellitus via molecular events of bacterial DPP4 activities.

Bacterial protease uses distinct thermodynamic signatures for substrate recognition.

Porphyromonas gingivalis and Porphyromonas endodontalis are important bacteria related to periodontitis, the most common chronic inflammatory disease in humans worldwide. Its comorbidity with systemic diseases, such as type 2 diabetes, oral cancers and cardiovascular diseases, continues to generate considerable interest. Surprisingly, these two microorganisms do not ferment carbohydrates; rather they use proteinaceous substrates as carbon and energy sources. However, the underlying biochemical mechanisms of their energy metabolism remain unknown. Here, we show that dipeptidyl peptidase 11 (DPP11), a central metabolic enzyme in these bacteria, undergoes a conformational change upon peptide binding to distinguish substrates from end products. It binds substrates through an entropy-driven process and end products in an enthalpy-driven fashion. We show that increase in protein conformational entropy is the main-driving force for substrate binding via the unfolding of specific regions of the enzyme ("entropy reservoirs"). The relationship between our structural and thermodynamics data yields a distinct model for protein-protein interactions where protein conformational entropy modulates the binding free-energy. Further, our findings provide a framework for the structure-based design of specific DPP11 inhibitors.

Dominant prevalence of Porphyromonas gingivalis fimA types I and IV in healthy Japanese children.

BACKGROUND/PURPOSE: Porphyromonas gingivalis is a major causative agent of chronic periodontitis, whilst circumstances for acquisition of the bacterium remain to be elucidated. To examine prevalence of the bacterium harboring distinct fimA types in dental plaque of children, we established PCR procedures that are applicable for specimens with limited amounts. By this method, all six fimA types including type I and Ib were directly identified, and prevalence of fimA types and their frequency of guardian-child transmission in Japanese children were assessed. MATERIALS AND METHODS: Genomic DNA was purified from dental plaque specimens of 132 periodontally healthy children (2-12 years old, 4.8 ± 0.2 years) and 19 mothers of resultant P. gingivalis-positive child subjects. PCR-based fimA genotyping was performed, and untypeable strains in the first PCR analysis were determined by a nested PCR. RESULTS: P. gingivalis was found in 15.2% of the subjects (2-10 years old, 5.1 ± 0.6 years), and the most prevalent types were I and IV (37.0% each), followed by Ib and III (11.1% each), and then II (7.4%). Seven (35.0%) of the 20 P. gingivalis-positive subjects had combined colonization of type I with other fimA types. In most cases, bacterial prevalence and fimA types in the children were distinct from those of their mothers, indicating that its maternal transmission was not significant. CONCLUSION: These results suggest that colonization of non-disease-associated fimA types I and IV P. gingivalis to the oral cavity initiates from early childhood without showing any periodontal inflammation.

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

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) (ß-propeller domain); and residues Ala(496)-Phe(736) (α/ß-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.

Exopeptidases and gingipains in Porphyromonas gingivalis as prerequisites for its amino acid metabolism.

Porphyromonas gingivalis, an asaccharolytic bacterium, utilizes amino acids as energy and carbon sources. Since amino acids are incorporated into the bacterial cells mainly as di- and tri-peptides, exopeptidases including dipeptidyl-peptidase (DPP) and tripeptidyl-peptidase are considered to be prerequisite components for their metabolism. We recently discovered DPP11, DPP5, and acylpeptidyl oligopeptidase in addition to previously reported DPP4, DPP7, and prolyl tripeptidyl peptidase A. DPP11 is a novel enzyme specific for acidic P1 residues (Asp and Glu) and distributed ubiquitously in eubacteria, while DPP5 is preferential for the hydrophobic P1 residue and the first entity identified in prokaryotes. Recently, acylpeptidyl oligopeptidase with a preference for hydrophobic P1 residues was found to release N-terminally blocked di- and tri-peptides. Furthermore, we also demonstrated that gingipains R and K contribute to P1-basic dipeptide production. These observations implicate that most, if not all, combinations of di- and tri-peptides are produced from extracellular oligopeptides even with an N-terminal modification. Here, we review P. gingivalis exopeptidases mainly in regard to their enzymatic characteristics. These exopeptidases with various substrate specificities benefit P. gingivalis for obtaining energy and carbon sources from the nutritionally limited subgingival environment.

Identification of Dipeptidyl-Peptidase (DPP)5 and DPP7 in Porphyromonas endodontalis, Distinct from Those in Porphyromonas gingivalis.

Dipeptidyl peptidases (DPPs) that liberate dipeptides from the N-terminal end of oligopeptides are crucial for the growth of Porphyromonas species, anaerobic asaccharolytic gram negative rods that utilize amino acids as energy sources. Porphyromonas endodontalis is a causative agent of periapical lesions with acute symptoms and Asp/Glu-specific DPP11 has been solely characterized in this organism. In this study, we identified and characterized two P. endodontalis DPPs, DPP5 and DPP7. Cell-associated DPP activity toward Lys-Ala-4-methylcoumaryl-7-amide (MCA) was prominent in P. endodontalis ATCC 35406 as compared with the Porphyromonas gingivalis strains ATCC 33277, 16-1, HW24D1, ATCC 49417, W83, W50, and HNA99. The level of hydrolysis of Leu-Asp-MCA by DPP11, Gly-Pro-MCA by DPP4, and Met-Leu-MCA was also higher than in the P. gingivalis strains. MER236725 and MER278904 are P. endodontalis proteins belong to the S9- and S46-family peptidases, respectively. Recombinant MER236725 exhibited enzymatic properties including substrate specificity, and salt- and pH-dependence similar to P. gingivalis DPP5 belonging to the S9 family. However, the kcat/Km figure (194 µM-1·sec-1) for the most potent substrate (Lys-Ala-MCA) was 18.4-fold higher as compared to the P. gingivalis entity (10.5 µM-1·sec-1). In addition, P. endodontalis DPP5 mRNA and protein contents were increased several fold as compared with those in P. gingivalis. Recombinant MER278904 preferentially hydrolyzed Met-Leu-MCA and exhibited a substrate specificity similar to P. gingivalis DPP7 belonging to the S46 family. In accord with the deduced molecular mass of 818 amino acids, a 105-kDa band was immunologically detected, indicating that P. endodontalis DPP7 is an exceptionally large molecule in the DPP7/DPP11/S46 peptidase family. The enhancement of four DPP activities was conclusively demonstrated in P. endodontalis, and remarkable Lys-Ala-MCA-hydrolysis was achieved by qualitative and quantitative potentiation of the DPP5 molecule.

Identification and characterization of prokaryotic dipeptidyl-peptidase 5 from Porphyromonas gingivalis.

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.

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

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 k cat/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 k cat 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.

Prediction of periodontopathic bacteria in dental plaque of periodontal healthy subjects by measurement of volatile sulfur compounds in mouth air.

OBJECTIVES: The aim of this study was to determine whether measurements of volatile sulfur compounds (VSCs) are useful to predict colonization of periodontopathic bacteria. For this purpose, we assessed the relationships among distributions of 4 species of periodontopathic bacteria in tongue coating and dental plaque, oral conditions including VSC concentration in mouth air, and smoking habit of periodontal healthy young subjects. METHODS: The subjects were 108 young adults (mean age, 23.5±2.56 years) without clinical periodontal pockets. Information regarding smoking habit was obtained by interview. After VSC concentration in mouth, air was measured with a portable sulfide monitor (Halimeter(®)), non-stimulated saliva flow and dental caries status were assessed, and tongue coating and dental plaque samples were collected from the subjects. The tongue coating samples were weighed to determine the amount. The colonization of Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Treponema denticola in both tongue coating and plaque samples was investigated using species-specific polymerase chain reaction assays. RESULTS: Significant relationships were observed between the colonization of periodontopathic bacteria in tongue coating and plaque samples, especially that of P. gingivalis. VSC concentration showed the most significant association with colonization of P. gingivalis in both tongue coating and dental plaque. Logistic regression analysis demonstrated that the adjusted partial correlation coefficient [Exp(B)] values for VSC concentration with the colonization of P. gingivalis, P. intermedia, and T. denticola in dental plaque were 135, 35.4 and 10.4, respectively. In addition, smoking habit was also shown to be a significant variable in regression models [Exp(B)=6.19, 8.92 and 2.53, respectively]. Therefore, receiver operating characteristic analysis was performed to predict the colonization of periodontal bacteria in dental plaque in the subjects divided by smoking habit. Based on our results, we found cut-off values that indicated likelihood ratios (LR) within the efficient range for positive findings in both groups. CONCLUSION: The present results demonstrated that measurement of VSC concentration in mouth air is a useful method to predict the presence of colonization of some periodontopathic bacteria in dental plaque.