CPG70 is a novel basic metallocarboxypeptidase with C-terminal polycystic kidney disease domains from Porphyromonas gingivalis.
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.
Chen
Yu-Yen
YY
School of Dental Science, University of Melbourne, 711 Elizabeth Street, Melbourne, Victoria 3000, Australia.
Cross
Keith J
KJ
Paolini
Rita A
RA
Fielding
James E
JE
Slakeski
Nada
N
Reynolds
Eric C
EC
D000595:Amino Acid Sequence; D000818:Animals; D002268:Carboxypeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000633:toxicity; D020134:Catalytic Domain; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D007690:Polycystic Kidney Diseases / Q000209:etiology*; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000472:pathogenicity
D002268:Carboxypeptidases