Gene_locus Report for: porgi-DPP

The Gram-negative anaerobe Porphyromonas gingivalis is associated with chronic periodontitis. Clinical isolates of P. gingivalis strains with high dipeptidyl peptidase 4 (DPP4) expression also had a high capacity for biofilm formation and were more infective. The X-ray crystal structure of P. gingivalis DPP4 was solved at 2.2 A resolution. Despite a sequence identity of 32%, the overall structure of the dimer was conserved between P. gingivalis DPP4 and mammalian orthologues. The structures of the substrate binding sites were also conserved, except for the region called S2-extensive, which is exploited by specific human DPP4 inhibitors currently used as antidiabetic drugs. Screening of a collection of 450 compounds as inhibitors revealed a structure-activity relationship that mimics in part that of mammalian DPP9. The functional similarity between human and bacterial DPP4 was confirmed using 124 potential peptide substrates.

Porphyromonas gingivalis is a major pathogen associated with adult periodontitis. We cloned and sequenced the gene (dpp) coding for dipeptidyl aminopeptidase IV (DPPIV) from P. gingivalis W83, based on the amino acid sequences of peptide fragments derived from purified DPPIV. An Escherichia coli strain overproducing P. gingivalis DPPIV was constructed. The enzymatic properties of recombinant DPPIV purified from the overproducer were similar to those of DPPIV isolated from P. gingivalis. The three amino acid residues Ser, Asp, and His, which are thought to form a catalytic triad in the C-terminal catalytic domain of eukaryotic DPPIV, are conserved in P. gingivalis DPPIV. When each of the corresponding residues of the enzyme was substituted with Ala by site-directed mutagenesis, DPPIV activity significantly decreased, suggesting that these three residues of P. gingivalis DPPIV are involved in the catalytic reaction. DPPIV-deficient mutants of P. gingivalis were constructed and subjected to animal experiments. Mice injected with the wild-type strain developed abscesses to a greater extent and died more frequently than those challenged with mutant strains. Mice injected with the mutants exhibited faster recovery from the infection, as assessed by weight gain and the rate of lesion healing. This decreased virulence of mutants compared with the parent strain suggests that DPPIV is a potential virulence factor of P. gingivalis and may play important roles in the pathogenesis of adult periodontitis induced by the organism.

We previously constructed a Porphyromonas gingivalis genomic library and isolated the 2.9 kb EcoRV fragment which specified glycylprolyl dipeptidyl aminopeptidase (GPase). Nucleotide sequencing of this fragment identified the single 2169 bp open reading frame which coded for a 723 amino acid protein. The amino acid sequencing of the NH2-terminal domain of the native and recombinant mature enzymes suggested that the protease possessed a 16 amino acid residue signal peptide. The calculated mass of the precursor and mature proteases were 82,018 and 80,235 daltons, respectively. The homology search of this enzyme in registered protein sequences revealed that this enzyme was homologous to dipeptidyl peptidase (DPP) IV from the Flavobacterium meningosepticum and that from eukaryotic cells, including the human, mouse, and rat. Three amino acid residues, Ser-593, Asp-668, and His-700, were identified as a putative catalytic triad, a common feature of eukaryotic serine proteases. In addition, this enzyme showed a broad proteolytic spectrum toward synthetic substrates capable of splitting not only Gly-Pro-derivative but also Ala-Pro, Lys-Pro, and Phe-Pro-derivatives. Therefore, we conclude that this enzyme belongs to DPP IV rather than GPase.

The Gram-negative anaerobe Porphyromonas gingivalis is associated with chronic periodontitis. Clinical isolates of P. gingivalis strains with high dipeptidyl peptidase 4 (DPP4) expression also had a high capacity for biofilm formation and were more infective. The X-ray crystal structure of P. gingivalis DPP4 was solved at 2.2 A resolution. Despite a sequence identity of 32%, the overall structure of the dimer was conserved between P. gingivalis DPP4 and mammalian orthologues. The structures of the substrate binding sites were also conserved, except for the region called S2-extensive, which is exploited by specific human DPP4 inhibitors currently used as antidiabetic drugs. Screening of a collection of 450 compounds as inhibitors revealed a structure-activity relationship that mimics in part that of mammalian DPP9. The functional similarity between human and bacterial DPP4 was confirmed using 124 potential peptide substrates.

The gram-negative anaerobic bacterium Porphyromonas gingivalis is a major causative agent of chronic periodontitis. Porphyromonas gingivalis strains have been classified into virulent and less-virulent strains by mouse subcutaneous soft tissue abscess model analysis. Here, we present the whole genome sequence of P. gingivalis ATCC 33277, which is classified as a less-virulent strain. We identified 2090 protein-coding sequences (CDSs), 4 RNA operons, and 53 tRNA genes in the ATCC 33277 genome. By genomic comparison with the virulent strain W83, we identified 461 ATCC 33277-specific and 415 W83-specific CDSs. Extensive genomic rearrangements were observed between the two strains: 175 regions in which genomic rearrangements have occurred were identified. Thirty-five of those genomic rearrangements were inversion or translocation and 140 were simple insertion, deletion, or replacement. Both strains contained large numbers of mobile elements, such as insertion sequences, miniature inverted-repeat transposable elements (MITEs), and conjugative transposons, which are frequently associated with genomic rearrangements. These findings indicate that the mobile genetic elements have been deeply involved in the extensive genome rearrangement of P. gingivalis and the occurrence of many of the strain-specific CDSs. We also describe here a very unique feature of MITE400, which we renamed MITEPgRS (MITE of P. gingivalis with Repeating Sequences).

The asaccharolytic periodontopathogen Porphyromonas gingivalis produces membrane-anchored proteases such as dipeptidyl peptidase IV that are involved in the destruction of host periodontal tissue. The extracellular domain of this enzyme was overexpressed in Escherichia coli as an N-terminal His-tag fusion protein, purified using standard metal-affinity chromatography and crystallized using the hanging-drop vapour-diffusion technique in 40% 2-methyl-2,4-pentanediol and 100 mM Tris-HCl pH 8.0. Diffraction data to 2.7 A resolution were collected using synchrotron radiation. The crystals belong to space group P2(1), with unit-cell parameters a = 117.0, b = 112.9, c = 310.0 A, beta = 95.0 degrees. There are ten molecules per asymmetric unit, indicating a solvent content of 50%. Data were also collected from selenomethionine-derived crystals and structure solution by SAD or MAD is in progress.

The complete 2,343,479-bp genome sequence of the gram-negative, pathogenic oral bacterium Porphyromonas gingivalis strain W83, a major contributor to periodontal disease, was determined. Whole-genome comparative analysis with other available complete genome sequences confirms the close relationship between the Cytophaga-Flavobacteria-Bacteroides (CFB) phylum and the green-sulfur bacteria. Within the CFB phyla, the genomes most similar to that of P. gingivalis are those of Bacteroides thetaiotaomicron and B. fragilis. Outside of the CFB phyla the most similar genome to P. gingivalis is that of Chlorobium tepidum, supporting the previous phylogenetic studies that indicated that the Chlorobia and CFB phyla are related, albeit distantly. Genome analysis of strain W83 reveals a range of pathways and virulence determinants that relate to the novel biology of this oral pathogen. Among these determinants are at least six putative hemagglutinin-like genes and 36 previously unidentified peptidases. Genome analysis also reveals that P. gingivalis can metabolize a range of amino acids and generate a number of metabolic end products that are toxic to the human host or human gingival tissue and contribute to the development of periodontal disease.

Porphyromonas gingivalis is a major pathogen associated with adult periodontitis. We cloned and sequenced the gene (dpp) coding for dipeptidyl aminopeptidase IV (DPPIV) from P. gingivalis W83, based on the amino acid sequences of peptide fragments derived from purified DPPIV. An Escherichia coli strain overproducing P. gingivalis DPPIV was constructed. The enzymatic properties of recombinant DPPIV purified from the overproducer were similar to those of DPPIV isolated from P. gingivalis. The three amino acid residues Ser, Asp, and His, which are thought to form a catalytic triad in the C-terminal catalytic domain of eukaryotic DPPIV, are conserved in P. gingivalis DPPIV. When each of the corresponding residues of the enzyme was substituted with Ala by site-directed mutagenesis, DPPIV activity significantly decreased, suggesting that these three residues of P. gingivalis DPPIV are involved in the catalytic reaction. DPPIV-deficient mutants of P. gingivalis were constructed and subjected to animal experiments. Mice injected with the wild-type strain developed abscesses to a greater extent and died more frequently than those challenged with mutant strains. Mice injected with the mutants exhibited faster recovery from the infection, as assessed by weight gain and the rate of lesion healing. This decreased virulence of mutants compared with the parent strain suggests that DPPIV is a potential virulence factor of P. gingivalis and may play important roles in the pathogenesis of adult periodontitis induced by the organism.

We previously constructed a Porphyromonas gingivalis genomic library and isolated the 2.9 kb EcoRV fragment which specified glycylprolyl dipeptidyl aminopeptidase (GPase). Nucleotide sequencing of this fragment identified the single 2169 bp open reading frame which coded for a 723 amino acid protein. The amino acid sequencing of the NH2-terminal domain of the native and recombinant mature enzymes suggested that the protease possessed a 16 amino acid residue signal peptide. The calculated mass of the precursor and mature proteases were 82,018 and 80,235 daltons, respectively. The homology search of this enzyme in registered protein sequences revealed that this enzyme was homologous to dipeptidyl peptidase (DPP) IV from the Flavobacterium meningosepticum and that from eukaryotic cells, including the human, mouse, and rat. Three amino acid residues, Ser-593, Asp-668, and His-700, were identified as a putative catalytic triad, a common feature of eukaryotic serine proteases. In addition, this enzyme showed a broad proteolytic spectrum toward synthetic substrates capable of splitting not only Gly-Pro-derivative but also Ala-Pro, Lys-Pro, and Phe-Pro-derivatives. Therefore, we conclude that this enzyme belongs to DPP IV rather than GPase.