Yano T, Fukamachi H, Yamamoto M, Igarashi T
Department of Periodontology, Showa University School of Dentistry, Shinagawa-ku, Tokyo, Japan.
Oral Microbiol Immunol. 2009 Dec;24(6):485-92. doi: 10.1111/j.1399-302X.2009.00546.x.
Hydrogen sulfide is responsible for lysis of red blood cells and is a major compound for oral malodor. To clarify the production mechanism of hydrogen sulfide in Prevotella intermedia, we found an L-cysteine desulfhydrase gene (lcs) homologue on the genome database of P. intermedia ATCC25611 and characterized its gene product.
The lcs gene homologue cloned into pGEX6p-1 vector was expressed in Escherichia coli and purified. Lcs activity was assayed by detection of the reaction products (hydrogen sulfide and pyruvate) or its derivatives from L-cysteine. Site-directed mutagenesis was used to convert an amino acid of the Lcs molecule.
The purified lcs gene product catalysed the degradation of L-cysteine to pyruvate, ammonia, and hydrogen sulfide, indicating that the protein is L-cysteine desulfhydrase. The enzyme required pyridoxal 5'-phosphate as a cofactor, and it was highly active at pH 7.0 and completely inhibited by ZnCl(2). The K(m) and V(max) of the enzyme were 0.7 mm and 4.2 micromol/min/mg, respectively. Replacement of Tyr-59, Tyr-118, Asp-198, and Lys-233 with any of the amino acids resulted in the complete disappearance of Lcs activity, implying that these amino acids are essential for enzyme activity. In addition, hydrogen sulfide produced by this enzyme lysed sheep red blood cells and modified hemoglobin.
These results show the enzymatic properties of L-cysteine desulfhydrase from P. intermedia ATCC25611 and also suggest that the Lcs enzyme, which produces hydrogen sulfide from L-cysteine, is closely associated with the pathogenesis of P. intermedia.
硫化氢可导致红细胞溶解，是口腔异味的主要成分。为阐明中间普氏菌中硫化氢的产生机制，我们在中间普氏菌ATCC25611的基因组数据库中发现了一个L-半胱氨酸脱硫酶基因（lcs）同源物，并对其基因产物进行了表征。
将克隆到pGEX6p-1载体中的lcs基因同源物在大肠杆菌中表达并纯化。通过检测L-半胱氨酸的反应产物（硫化氢和丙酮酸）或其衍生物来测定Lcs活性。使用定点诱变来改变Lcs分子的一个氨基酸。
纯化的lcs基因产物催化L-半胱氨酸降解为丙酮酸、氨和硫化氢，表明该蛋白质是L-半胱氨酸脱硫酶。该酶需要磷酸吡哆醛作为辅因子，在pH 7.0时具有高活性，并被ZnCl₂完全抑制。该酶的K(m)和V(max)分别为0.7 mM和4.2 μmol/min/mg。用任何一种氨基酸替代Tyr-59、Tyr-118、Asp-198和Lys-233都会导致Lcs活性完全消失，这意味着这些氨基酸对酶活性至关重要。此外，该酶产生的硫化氢可溶解绵羊红细胞并修饰血红蛋白。
这些结果显示了中间普氏菌ATCC25611中L-半胱氨酸脱硫酶的酶学性质，也表明从L-半胱氨酸产生硫化氢的Lcs酶与中间普氏菌的发病机制密切相关。
