Study of membrane transport system in proteinase produced from Porphyromonas gingivalis

赖氨酸特异性半胱氨酸蛋白酶（Lys-gingipain，Kgp）是由牙龈卟啉单胞菌（Porphyromonas gingivalis）产生的一种独特的半胱氨酸蛋白酶，与牙龈卟啉单胞菌的致病性密切相关。然而，由于难以获得足够量的酶，对其结构和功能特性的研究受到限制。在这里，我们显示了催化活性的重组Kgp在牙龈卟啉单胞菌Kgp无效突变体中的表达，并通过使用稳定存在于牙龈卟啉单胞菌中的穿梭质粒载体恢复其功能。表达Kgp的突变体表现出与野生型菌株相似的催化活性。该突变体还恢复了在血琼脂平板上形成黑色着色菌落的能力，并产生负责血红蛋白结合的19 kDa血红蛋白受体蛋白。此外，为了确定活性位点Cys残基及其在细菌黑色色素沉着中的重要性，我们构建了三个具有改变的潜在活性位点Cys残基的Kgp突变体。表达单一突变体（C476 A）的细胞显示高Kgp活性和黑色色素沉着。相反，表达单突变体（C477 A）和双突变体（C476 A/C477 A）的细胞既不显示Kgp活性，也不显示黑色色素沉着。结果表明，477^<th>Cys残基是Kgp活性和牙龈卟啉单胞菌黑色色素形成所必需的。

A lysine-specific cysteine proteinase (Lys-gingipain, Kgp) is a unique cysteine proteinase produced by Porphyromonas gingivalis, a major etiological bacterium of periodontal diseases, and is implicated in the virulence of this bacterium. Studies on its structural and functional properties, however, have been limited by the difficulty in obtaining a sufficient amount of the enzyme. Here we show the expression of catalytically active recombinant Kgp in a P.gingivalis Kgp-null mutant and the restoration of its functions by use of a shuttle plasmid vector stably existing 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 hemoglobin receptor protein responsible for hemoglobin binding. Furthermore, to establish the active-site Cys residue and its importance in the bacterial black pigmentation, we constructed three Kgp mutants with changed potential active-site Cys residues. The cells expressing single mutant (C476A) showed the high Kgp activity and the black pigmentation. By contrast, the cells expressing single mutant (C477A) and the double mutant (C476A/C477A) exhibited neither Kgp activity nor black pigmentation. The results indicate that 477^<th> Cys residue is essential for Kgp activity and the black pigmentation of P.gingivalis.