Phenylalanine 664 of dipeptidyl peptidase (DPP) 7 and Phenylalanine 671 of DPP11 mediate preference for P2-position hydrophobic residues of a substrate (original) (raw)
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Biochimie, 2013
Porphyromonas gingivalis, an asaccharolytic gram-negative rod-shaped bacterium, expresses the novel Asp/Glu-specific dipeptidyl-peptidase (DPP) 11 (Ohara-Nemoto, Y. et al., (2011) J. Biol. Chem. 286, 38115-38127), which has been categorized as a member of the S46/DPP7 family that is preferential for hydrophobic residues at the P1 position. From that finding, 129 gene products constituting five clusters from the phylum Bacteroidetes have been newly annotated to either DPP7 or DPP11, whereas the remaining 135 members, mainly from the largest phylum Proteobacteria, have yet to be assigned. In this study, the substrate specificities of the five clusters and an unassigned group were determined with recombinant DPPs from typical species, i.e., P. gingivalis, Capnocytophaga gingivalis, Flavobacterium psychrophilum, Bacteroides fragilis, Bacteroides vulgatus, and Shewanella putrefaciens.
Biochimie, 2017
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, Gly(673) in DPP7 and Arg(673) 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 Gly(673) 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...
Journal of Biological Chemistry, 2011
Background: Dipeptidyl peptidases (DPPs) are required for protein metabolism in Porphyromonas gingivalis. Results: Asp/Glu-specific novel DPP (DPP11) was discovered and characterized. Conclusion: DPP11 ensures efficient degradation of oligopeptide substrates in Gram-negative anaerobic rods. Significance: This observation suggests further variation of substrate specificity in the DPP members. Porphyromonas gingivalis and Porphyromonas endodontalis, asaccharolytic black-pigmented anaerobes, are predominant pathogens of human chronic and periapical periodontitis, respectively. They incorporate di-and tripeptides from the environment as carbon and energy sources. In the present study we cloned a novel dipeptidyl peptidase (DPP) gene of P. endodontalis ATCC 35406, designated as DPP11. The DPP11 gene encoded 717 amino acids with a molecular mass of 81,090 Da and was present as a 75-kDa form with an N terminus of Asp 22. A homology search revealed the presence of a P. gingivalis orthologue, PGN0607, that has been categorized as an isoform of authentic DPP7. P. gingivalis DPP11 was exclusively cell-associated as a truncated 60-kDa form, and the gene ablation retarded cell growth. DPP11 specifically removed dipeptides from oligopeptides with the penultimate N-terminal Asp and Glu and has a P2-position preference to hydrophobic residues. Optimum pH was 7.0, and the k cat /K m value was higher for Asp than Glu. Those activities were lost by substitution of Ser 652 in P. endodontalis and Ser 655 in P. gingivalis DPP11 to Ala, and they were consistently decreased with increasing NaCl concentration. Arg 670 is a unique amino acid completely conserved in all DPP11 members distributed in the genera Porphyromonas, Bacteroides, and Parabacteroides, whereas this residue is converted to Gly in all authentic DPP7 members. Substitution analysis suggested that Arg 670 interacts with an acidic residue of the substrate. Considered to preferentially utilize acidic amino acids, DPP11 ensures efficient degradation of oligopeptide substrates in these Gram-negative anaerobic rods. Porphyromonas gingivalis, a Gram-negative black-pigmented anaerobe, is a major causative organism of aggressive forms of chronic periodontitis (1, 2) which leads to loss of permanent teeth (3-5). Recently, much attention has been paid to this bacterium because of its close relationship with systemic diseases, such as cardiovascular diseases (6), decreased kidney function (7), and rheumatoid arthritis (8). An important relative of the bacterium is Porphyromonas endodontalis, which is predominantly isolated from periapical periodontitis sites, i.e. infected root canals with acute symptoms such as pain, swelling, and drainage (9-11). Both Porphyromonas species are asaccharolytic in principal and do not ferment glucose, cellobiose, lactose, or sucrose (12) and require proteinaceous substrates as carbon and energy sources. P. gingivalis possesses extracellular arginine aminopeptidase activity, which is mediated by Arg-gingipains (Rgps) 3 isoforms A and B of Arg-X-specific cysteine proteinase (X is any amino acid), whereas other aminopeptidase activities are not present (13, 14). Lys-specific gingipain (Kgp), another potent cysteine proteinase, does not exhibit aminopeptidase activity (15). In accord with the lack of predominant aminopeptidase activities, it has been demonstrated that P. gingivalis mainly incorporates nutritional amino acids as forms of di-and tripeptides, not as single amino acids, and produces metabolic end products such as ammonia, acetate, propionate, and butyrate (16, 17), which are considered to be virulence factors of this bacterium, causing host tissue damage (18, 19). Accordingly, the cell-surface and extracellular peptidases of P. gingivalis that produce di-and tri-peptides are considered to play critical roles in cell growth as well as its pathogenicity. Although entire genome sequencing has annotated 72 peptidase genes in P. gingivalis W83 (20) as well as ATCC 33277 (21), * This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports,
Journal of Biological Chemistry, 2014
Background: Dipeptidyl peptidases (DPPs) are key factors for amino acid metabolism and bacterial growth of asaccharolytic Porphyromonas gingivalis. Results: DPP5, which is specific for Ala and hydrophobic residues, is expressed in the periplasmic space of P. gingivalis. Conclusion: DPP5 was discovered in prokaryotes for the first time. Significance: The discovery of DPP5 expands understanding of amino acid and energy metabolism in prokaryotes.
PloS one, 2014
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 exhib...
Scientific Reports, 2015
The dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) belongs to the S46 family of serine peptidases and preferentially cleaves substrates with Asp/Glu at the P1 position. The molecular mechanism underlying the substrate specificity of PgDPP11, however, is unknown. Here, we report the crystal structure of PgDPP11. The enzyme contains a catalytic domain with a typical double β-barrel fold and a recently identified regulatory α-helical domain. Crystal structure analyses, docking studies, and biochemical studies revealed that the side chain of Arg673 in the S1 subsite is essential for recognition of the Asp/Glu side chain at the P1 position of the bound substrate. Because S46 peptidases are not found in mammals and the Arg673 is conserved among DPP11s, we anticipate that DPP11s could be utilised as targets for antibiotics. In addition, the present structure analyses could be useful templates for the design of specific inhibitors of DPP11s from pathogenic organisms. Periodontitis is a bacterially induced inflammatory disease that destroys the periodontal tissues, eventually leading to tooth loss 1. Periodontitis is widely regarded as the second most common disease worldwide, and chronic periodontitis affects approximately 750 million people as of 2010 2. Various studies have revealed that periodontitis is associated with systemic diseases such as diabetes and cardiovascular disease 3 , preterm and low-weight births 4 , Alzheimer's disease 5 , cancers 6 , respiratory diseases 7 and rheumatoid arthritis 8. Porphyromonas gingivalis, a Gram-negative, anaerobic bacterium, is a major pathogen associated with the chronic form of periodontitis 1,9. Because P. gingivalis is an asaccharolytic bacterium that gains its metabolic energy by fermenting amino acids 10 , P. gingivalis secretes various proteases/ peptidases that are capable of digesting external proteins into peptides. The best-characterised proteases from P. gingivalis are two cysteine proteases, gingipains R (Rgp) and K (Kgp), that exhibit specificity for arginine and lysine, respectively 11,12. Rgp and Kgp have been implicated as major virulence factors of P. gingivalis 13-15 , and inhibitors of these proteases have been reported to suppress the virulence of
Scientific Reports, 2018
Dipeptidyl peptidase IV (DPP IV, DPP4, or DAP IV) preferentially cleaves substrate peptides with Pro or Ala at the P1 position. The substrate recognition mechanism has been fully elucidated for mammalian DPP IV by crystal structure analyses but not for bacterial orthologues. Here, we report the crystal structures of a bacterial DPP IV (PmDAP IV) in its free form and in complexes with two kinds of dipeptides as well as with a non-peptidyl inhibitor at 1.90 to 2.47 Å resolution. Acyl-enzyme intermediates were observed for the dipeptide complexes of PmDAP IV, whereas tetrahedral intermediates were reported for the oligopeptide complexes of mammalian DPP IVs. This variation reflects the different structural environments of the active site Arg residues, which are involved in the recognition of a substrate carbonyl group, of mammalian and bacterial enzymes. A phylogenetic analysis revealed that PmDAP IV is a closer relative of dipeptidyl peptidases 8 and 9 (DPP8 and DPP9, DPP IV-family en...
Characterization of two M17 family members in Escherichia coli, Peptidase A and Peptidase B
Biochemical and Biophysical Research Communications, 2010
Comparative biochemistry M1 family peptidase M17 family peptidase Substrate specificity a b s t r a c t Escherichia coli encodes two aminopeptidases belonging to the M17 family: Peptidase A (PepA) and Peptidase B (PepB). To gain insights into their substrate specificities, PepA or PepB were overexpressed in DpepN, which shows greatly reduced activity against the majority of amino acid substrates. Overexpression of PepA or PepB increases catalytic activity of several aminopeptidase substrates and partially rescues growth of DpepN during nutritional downshift and high temperature stress. Purified PepA and PepB display broad substrate specificity and Leu, Lys, Met and Gly are preferred substrates. However, distinct differences are observed between these two paralogs: PepA is more stable at high temperature whereas PepB displays broader substrate specificity as it cleaves Asp and insulin B chain peptide. Importantly, this strategy, i.e. overexpression of peptidases in DpepN and screening a panel of substrates for cleavage, can be used to rapidly identify peptidases with novel substrate specificities encoded in genomes of different organisms.
A statistical investigation of amphiphilic properties of C-terminally anchored peptidases
European Biophysics Journal, 2003
A number of DD-peptidases have been reported to interact with the membrane via C-terminal amphiphilic a-helices, but experimental support for this rests with a few well-characterized cases. These show the C-terminal interactions of DD-carboxypeptidases to involve high levels of membrane penetration, DDendopeptidases to involve membrane surface binding and class C penicillin-binding proteins to involve membrane binding with intermediate properties. Here, we have characterized C-terminal a-helices from each of these peptidase groups according to their amphiphilicity, as measured by mean , and the corresponding mean hydrophobicity, . Regression and statistical analyses showed these properties to exhibit parallel negative linear relationships, which resulted from the spatial ordering of a-helix amino acid residues. Taken with the results of compositional and graphical analyses, our results suggest that the use of C-terminal a-helices may be a universal feature of the membrane anchoring for each of these groups of DD-peptidases. Moreover, to accommodate differences between these mechanisms, each group of C-terminal a-helices optimizes its structural amphiphilicity and hydrophobicity to fulfil its individual membrane-anchoring function. Our results also show that each anchor type analysed requires a similar overall balance between amphiphilicity for membrane interaction, which we propose is necessary to stabilize their initial membrane associations. In addition, we present a methodology for the prediction of C-terminal a-helical anchors from the classes of DD-peptidases analysed, based on a parallel linear model.
Biochimie, 2009
Enzymes belonging to the M1 family play important cellular roles and the key amino acids (aa) in the catalytic domain are conserved. However, C-terminal domain aa are highly variable and demonstrate distinct differences in organization. To address a functional role for the C-terminal domain, progressive deletions were generated in Tricorn interacting factor F2 from Thermoplasma acidophilum (F2) and Peptidase N from Escherichia coli (PepN). Catalytic activity was partially reduced in PepN lacking 4 C-terminal residues (PepNDC4) whereas it was greatly reduced in F2 lacking 10 C-terminal residues (F2DC10) or PepN lacking eleven C-terminal residues (PepNDC11). Notably, expression of PepNDC4, but not PepNDC11, in E. coliDpepN increased its ability to resist nutritional and high temperature stress, demonstrating physiological significance. Purified C-terminal deleted proteins demonstrated greater sensitivity to trypsin and bound stronger to 8-amino 1-napthalene sulphonic acid (ANS), revealing greater numbers of surface exposed hydrophobic aa. Also, F2 or PepN containing large aa deletions in the C-termini, but not smaller deletions, were present in high amounts in the insoluble fraction of cell extracts probably due to reduced protein solubility. Modeling studies, using the crystal structure of E. coli PepN, demonstrated increase in hydrophobic surface area and change in accessibility of several aa from buried to exposed upon deletion of C-terminal aa. Together, these studies revealed that non-conserved distal C-terminal aa repress the surface exposure of apolar aa, enhance protein solubility, and catalytic activity in two soluble and distinct members of the M1 family.