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首页> 外文期刊>Infection and immunity >Role for Recombinant γ-Glutamyltransferase from Treponema denticola in Glutathione Metabolism
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Role for Recombinant γ-Glutamyltransferase from Treponema denticola in Glutathione Metabolism

机译：密闭螺旋体中重组γ-谷氨酰转移酶在谷胱甘肽代谢中的作用

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Volatile sulfur compounds, including hydrogen sulfide (H2S), have been implicated in the development of periodontal disease. Glutathione is an important thiol source for H2S production in periodontal pockets. Our recent studies have delineated a pathway of glutathione metabolism in Treponema denticola that releases H2S. In this pathway, γ-glutamyltransferase (GGT) has been proposed to catalyze the first step of glutathione degradation. We have cloned the gene of GGT from T. denticola, which contains an open reading frame of 726 bp encoding a protein of 241 amino acids. Transformation of this gene into Escherichia coli led to the expression of a recombinant protein. After purification by chromatography, the recombinant protein showed enzymatic activity typical of GGT, catalyzing the degradation of Na-γ-glutamyl-4-nitroaniline (GNA) and the hydrolysis of glutathione, releasing glutamic acid or glutamine and cysteinylglycine. l-Cysteine is not a substrate of GGT. Importantly, GNA, when added to T. denticola, was able to compete with glutathione and inhibit the production of H2S, ammonia, and pyruvate. This was accompanied by the suppression of hemoxidative and hemolytic activities of the bacteria. Purified GGT was inactivated by TLCK (Nα-p-tosyl-l-lysine chloromethyl ketone) and proteinase K treatment. However, higher enzymatic activity was demonstrated in the presence of 2-mercaptoethanol and dithiothreitol. Our further experiments showed that the addition of recombinant GGT to Porphyromonas gingivalis, a bacterium without significant glutathione-metabolizing capacity, drastically increased the utilization of glutathione by the bacterium, producing H2S, ammonia, and pyruvate. This was again accompanied by enhanced bacterial hemoxidative and hemolytic activities. Together, the results suggest an important role for GGT in glutathione metabolism in oral bacteria.
机译：挥发性的硫化物，包括硫化氢（H 2 S），已与牙周疾病的发展有关。谷胱甘肽是牙周袋中H 2 S产生的重要硫醇来源。我们最近的研究描述了 Treponema denticola 中谷胱甘肽的代谢途径，该途径释放H 2 S。在该途径中，已经提出了γ-谷氨酰转移酶（GGT）来催化谷胱甘肽降解的第一步。我们已经从 T克隆了GGT基因。 denticola ，其中包含一个726 bp的开放阅读框，编码241个氨基酸。该基因转化到大肠埃希氏菌中导致重组蛋白的表达。经色谱纯化后，重组蛋白显示出典型的GGT酶活性，催化Na-γ-谷氨酰-4-硝基苯胺（GNA）的降解和谷胱甘肽的水解，释放出谷氨酸或谷氨酰胺和半胱氨酰甘氨酸。半胱氨酸不是GGT的底物。重要的是，将GNA添加到 T中时。能够与谷胱甘肽竞争并抑制H 2 S，氨和丙酮酸的产生。这伴随着细菌的半氧化和溶血活性的抑制。纯化的GGT通过TLCK（ N α- p -甲苯磺酰基-1-赖氨酸氯甲基酮）和蛋白酶K处理而失活。然而，在2-巯基乙醇和二硫苏糖醇的存在下证明了更高的酶活性。我们进一步的实验表明，向没有明显的谷胱甘肽代谢能力的细菌 Gorphyromonas gingivalis 中添加重组GGT可以大大提高细菌对谷胱甘肽的利用，从而产生H 2 S，氨水和丙酮酸。这又伴随着增强的细菌半氧化和溶血活性。在一起，结果表明GGT在口腔细菌中的谷胱甘肽代谢中具有重要作用。

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