title: Determination of active site of lysine-specific cysteine proteinase (Lys-gingipain) by use of a Porphyromonas gingivalis plasmid system.
abstract: 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.
authors.affiliation: Department of Oral Pathopharmacology, Unit of Basic Medical Sciences, Medical and Dental Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Ishida
authors.forename: Jinping
authors.initials: J
authors.lastname: Hu
authors.forename: Eiko
authors.initials: E
authors.lastname: Sakai
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kadowaki
authors.forename: Kenji
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Takayuki
authors.initials: T
authors.lastname: Tsukuba
authors.forename: Yuzo
authors.initials: Y
authors.lastname: Kato
authors.forename: Koji
authors.initials: K
authors.lastname: Nakayama
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Okamoto
mesh_terms: 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
chemical_list: D018829:Adhesins, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; D003546:Cysteine Endopeptidases