Abstract
A novel dipeptidylpeptidase (DPP-7) was purified from the membrane fraction of Porphyromonas gingivalis. This enzyme, with an apparent molecular mass of 76 kDa, has the specificity for both aliphatic and aromatic residues in the P1 position. Although it belongs to the serine class of peptidases, it does not resemble other known dipeptidylpeptidases. Interestingly, the amino acid sequence around the putative active site serine residue shows significant similarity to the C-terminal region of the Staphylococcus aureus V-8 endopeptidase. The genes encoding homologues of DPP-7 were found in genomes of Xylella fastidiosa, Shewanella putrefaciens, and P. gingivalis. It is likely that at least in P. gingivalis, DPP-7 and its homologue, in concert with other di- and tripeptidases, serve nutritional functions by providing dipeptides to this asaccharolytic bacterium.
Highlights
Tripeptidylpeptidase A, an enzyme that liberates tripeptides from the N-terminal regions of substrates containing proline residues in the third position
P. gingivalis cannot transport polyand oligopeptides into the cell, it has the ability to thrive on dipeptides as a sole source of carbon
We have focused our attention on a specialized group of P. gingivalis peptidases capable of hydrolyzing oligopeptides to di- and tripeptides, which can be subsequently metabolized by this periodontopathogen
Summary
Source and Cultivation of Bacteria—P. gingivalis DPP-7 was purified from strain HG66, a kind gift of Dr Roland Arnold (University of North Carolina, Chapel Hill, NC). Bound proteins were eluted with a potassium phosphate gradient (20 –300 mM), and fractions (7 ml) were analyzed for amidolytic activity against H-Ala-Phe-pNA. The active fractions were saturated with 1 M ammonium sulfate and loaded onto a phenyl-Sepharose HP column (Amersham Pharmacia Biotech) equilibrated with 50 mM potassium phosphate, pH 7.0, containing 1 M ammonium sulfate. The abbreviations used are: DPP, dipeptidylpeptidase; MES, 4-morpholineethanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; pNA, p-nitroanilide; Suc-, succinyl-; Z-, benzyloxycarbonyl; contig, group of overlapping clones; ORF, open reading frame. Extensively dialyzed against 20 mM MES, pH 6.6, and applied onto a MonoS HR 5/5 FPLC (Amersham Pharmacia Biotech) column equilibrated with the same buffer.
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