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J Biol Chem. 2002 Jun 28;277(26):23433-40. Epub 2002 Apr 25.

CPG70 is a novel basic metallocarboxypeptidase with C-terminal polycystic kidney disease domains from Porphyromonas gingivalis.

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1
School of Dental Science, University of Melbourne, 711 Elizabeth Street, Melbourne, Victoria 3000, Australia.

Abstract

In a search for a basic carboxypeptidase that might work in concert with the major virulence factors, the Arg- and Lys-specific cysteine endoproteinases of Porphyromonas gingivalis, a novel 69.8-kDa metallocarboxypeptidase CPG70 was purified to apparent homogeneity from the culture fluid of P. gingivalis HG66. Carboxypeptidase activity was measured by matrix-assisted laser desorption ionization-mass spectrometry using peptide substrates derived from a tryptic digest of hemoglobin. CPG70 exhibited activity with peptides containing C-terminal Lys and Arg residues. The k(cat)/K(m) values for the hydrolysis of the synthetic dipeptides FA-Ala-Lys and FA-Ala-Arg by CPG70 were 99 and 56 mm(-1)s(-1), respectively. The enzyme activity was strongly inhibited by the Arg analog (2-guanidinoethylmercapto)succinic acid and 1,10-phenanthroline. High resolution inductively coupled plasma-mass spectrometry demonstrated that 1 mol of CPG70 was associated with 0.6 mol of zinc, 0.2 mol of nickel, and 0.2 mol of copper. A search of the P. gingivalis W83 genomic data base (TIGR) with the N-terminal amino acid sequence determined for CPG70 revealed that the enzyme is an N- and C-terminally truncated form of a predicted 91.5-kDa protein (PG0232). Analysis of the deduced amino acid sequence of the full-length protein revealed an N-terminal signal sequence followed by a pro-segment, a metallocarboxypeptidase catalytic domain, three tandem polycystic kidney disease domains, and an 88-residue C-terminal segment. The catalytic domain exhibited the highest sequence identity with the duck metallocarboxypeptidase D domain II. Insertional inactivation of the gene encoding CPG70 resulted in a P. gingivalis isogenic mutant that was avirulent in the murine lesion model under the conditions tested.

PMID:
11976326
DOI:
10.1074/jbc.M200811200
[Indexed for MEDLINE]
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