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

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 Gramnegative 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 ? 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 amelanocyte-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

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 biofilm from adults with chronic periodontitis. Peptidases in periodontopathic bacteria are considered to function as etiological reagents. Pre. 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 fulllength 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

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

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

Single Nucleotide Polymorphism that Accompanies a Missense Mutation (Gln488His) Impedes the Dimerization of Hsp90.

A single nucleotide polymorphism (SNP) that causes a missense mutation of highly conserved Gln488 to His of the alpha isoform of the 90-kDa heat shock protein (Hsp90alpha) molecular chaperone is observed in Caucasians. The mutated Hsp90alpha severely reduced the growth of yeast cells. To investigate this molecular mechanism, we examined the domain-domain interactions of human Hsp90alpha by using bacterial 2-hybrid system. Hsp90alpha was expressed as a full-length form, N-terminal domain (residues 1-400), or middle (residues 401-617) plus C-terminal (residues 618-732) domains (MC domain/amino acids 401-732). The Gln488His substitution in MC domain did not affect the intra-molecular interaction with N-terminal domain, whereas the dimeric interaction-mediated by the inter-molecular interaction between MC domains was decreased to 32%. Gln488Ala caused a similar change, whereas Gln488Thr, which exceptionally occurs in mitochondrial Hsp90 paralog, fully maintained the dimeric interaction. Therefore, the SNP causing Gln488His mutation could abrogate the Hsp90 function due to reduced dimerization

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?±?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

A disulfide bridge mediated by cysteine 574 is formed in the dimer of the 70-kDa heat shock protein.

The 70-kDa heat shock protein (Hsp70) is predominantly present intracellularly as a monomer, but a small population is converted to dimers and oligomers under certain conditions. In the present study, we investigated the dimeric structure of human inducible Hsp70. As reported earlier, the C-terminal client-binding domain (amino acids 382-641) was required for the dimerization. A 40-amino acid deletion in the client-binding domain from either the N-terminus or C-terminus greatly enhanced the dimerization potential of Hsp70. Limited proteolysis indicated that the dimer formed through truncation from the C-terminus had a conformation similar to that of the non-truncated form. Truncation experiments demonstrated that the client-binding sub-domain (amino acids 382-520) with its adjacent region up to amino acid 541 was not sufficient for the dimerization but that the region up to amino acid 561 was sufficient. Interestingly, the dimer formed through truncation from the C-terminus acquired a homomeric disulfide bridge at Cys574