Cystalysin, a 46-kilodalton cysteine desulfhydrase from Treponema denticola, with hemolytic and hemoxidative activities
Cystalysin, a 46-kilodalton cysteine desulfhydrase from Treponema denticola, with hemolytic and hemoxidative activities
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DOI：
10.1128/iai.65.8.3231-3238.1997
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发表时间：
1997-08-01
影响因子：
3.1
通讯作者：
Holt, SC
中科院分区：
文献类型：
--
作者：
Chu, L;Ebersole, JL;Holt, SC
从密螺旋体（Treponema denticola） ATCC 35404中获得的46-kDa溶血蛋白（cystalysin）在大肠杆菌LC-67中过表达，纯化得到的46-kDa蛋白和重组46-kDa蛋白均表达相同的生物学和功能特性。除了具有裂解红细胞和将血红蛋白含氧氧化为高铁血红蛋白、溶胱氨酸的生物学功能外，还能够从特定的含硫化合物（如半胱氨酸）中去除巯基和氨基，产生H2S、NH3和丙酮酸。这种半胱氨酸脱硫酶产生以下Michaelis-Menten动力学：k -m = 3.6 mM, k(cat) = 12 s（-1）。半胱硫氨酸和s -氨基乙基- l-半胱氨酸也是该蛋白的底物。最终产物的气相色谱-质谱和高效液相色谱分析显示NH3、丙酮酸、同型半胱氨酸（来自半胱硫氨酸）和半胱氨酸（来自s -氨基乙基- l-半胱氨酸）。该酶在较宽的pH范围内具有活性，pH值为7.8 ~ 8.0时酶活性最高，β -巯基乙醇能提高酶活性。它不受蛋白酶抑制剂TLCK （N α -对- toyl -l -赖氨酸氯甲基酮）、pronase或蛋白酶K的抑制，这表明功能位点受到物理保护或位于多肽的一小段。我们假设cystalysin是一种含有吡哆醛-5-磷酸的酶，具有α C-N和β C-S裂解酶（胱氨酸酶）类型的活性，因为在深层牙周袋中有大量H2S的报道。囊溶素在体内也可能作为一种重要的毒力分子发挥作用。
A 46-kDa hemolytic protein, referred to as cystalysin, from Treponema denticola ATCC 35404 was overexpressed in Escherichia coli LC-67, Both the native and recombinant 46-kDa proteins were purified to homogeneity, Both proteins expressed identical biological and functional characteristics. In addition to its biological function of lysing erythrocytes and hemoxidizing the hemoglobin to methemoglobin, cystalysin,vas also capable of removing the sulfhydryl and amino groups from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate, This cysteine desulfhydrase resulted in the following Michaelis-Menten kinetics: K-m = 3.6 mM and k(cat) = 12 s(-1). Cystathionine and S-aminoethyl-L-cysteine were also substrates for the protein. Gas chromatography-mass spectrometry and high-performance liquid chromatography analysis of the end products revealed NH3, pyruvate, homocysteine (from cystathionine), and cysteamine (from S-aminoethyl-L-cysteine). The enzyme was active over a broad pH range, with highest activity at pH 7.8 to 8.0, The enzymatic activity was increased by beta-mercaptoethanol. It was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting that the functional site was physically protected or located in a small fragment of the polypeptide, We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme, with activity of an alpha C-N and beta C-S lyase (cystathionase) type, Since large amounts of H2S have been reported in deep periodontal pockets, cystalysin may also function in vivo as an important virulence molecule.