Determination of active site of lysine-specific cysteine proteinase (Lys-gingipain) by use of a Porphyromonas gingivalis plasmid system.
Porphyromonas gingivalis, a major etiological bacterium of periodontal diseases, produces a unique lysine-specific cysteine proteinase (Lys-gingipain, Kgp) implicated in the virulence of this organism. Our observations show the expression of a catalytically active recombinant Kgp in a P. gingivalis Kgp-null mutant and the restoration of its functions by the use of a shuttle plasmid vector stable in P. gingivalis. The Kgp-expressing mutant exhibited a similar catalytic activity to that of the wild-type strain. This mutant also restored the ability to form black-pigmented colonies on blood agar plates and to generate a 19-kDa haemoglobin receptor protein responsible for haemoglobin binding. In order to establish the importance of the active-site Cys residue and elucidate its role in bacterial black pigmentation we constructed three Kgp mutants with changed potential active-site Cys residues. The cells expressing a single mutation (C476A) showed the high Kgp activity and the black pigmentation. In contrast, the cells expressing the single mutant (C477A) and the double mutant (C476A/C477A) exhibited neither Kgp activity nor black pigmentation. These results indicate that the 477th Cys residue is essential for both the Kgp activity and the black pigmentation of P. gingivalis.
Ishida
Yutaka
Y
Department of Oral Pathopharmacology, Unit of Basic Medical Sciences, Medical and Dental Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan.
Hu
Jinping
J
Sakai
Eiko
E
Kadowaki
Tomoko
T
Yamamoto
Kenji
K
Tsukuba
Takayuki
T
Kato
Yuzo
Y
Nakayama
Koji
K
Okamoto
Kuniaki
K
D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D000080867:Gingipain Cysteine Endopeptidases; D006801:Humans; D009154:Mutation / Q000235:genetics; D010510:Periodontal Diseases / Q000201:enzymology* / Q000235:genetics; D010858:Pigmentation / Q000235:genetics; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000235:genetics
D018829:Adhesins, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; D003546:Cysteine Endopeptidases