Role for recombinant gamma-glutamyltransferase from Treponema denticola in glutathione metabolism.
Role for recombinant gamma-glutamyltransferase from Treponema denticola in glutathione metabolism.
复制标题
来自密螺旋体的重组γ-谷氨酰转移酶在谷胱甘肽代谢中的作用。
DOI：
10.1128/iai.71.1.335-342.2003
复制
发表时间：
2003
影响因子：
3.1
通讯作者：
Ebersole,JeffereyL
中科院分区：
文献类型：
--
作者：
Chu,Lianrui;Xu,Xiaoping;Dong,Zheng;Cappelli,David;Ebersole,JeffereyL
挥发性硫化合物，包括硫化氢（H2S），与牙周病的发展有关。谷胱甘肽是牙周袋中产生H2S的重要巯基来源。我们最近的研究描绘了一个谷胱甘肽代谢途径在密螺旋体denticola，释放H2S。在该途径中，γ-谷氨酰转移酶（GGT）被提议催化谷胱甘肽降解的第一步。我们从T中克隆了GGT基因。Denticola的开放阅读框为726 bp，编码241个氨基酸。将该基因转化大肠杆菌，获得了重组蛋白的表达。重组蛋白经层析纯化后，具有GGT的典型酶活性，可催化Na-γ-谷氨酰-4-硝基苯胺（GNA）的降解和谷胱甘肽的水解，释放谷氨酸或谷氨酰胺和半胱氨酰甘氨酸，L-半胱氨酸不是GGT的底物。重要的是，GNA，当添加到T。能够与谷胱甘肽竞争并抑制H2S、氨和丙酮酸的产生。这伴随着细菌的溶血和溶血活性的抑制。纯化的GGT经TLCK（Nα-对甲苯磺酰基-L-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，表现出更高的酶活性。我们进一步的实验表明，除了重组GGT toPorphyromonas gingivalis，细菌没有显着的谷胱甘肽代谢能力，大大增加了利用谷胱甘肽的细菌，产生H2S，氨，和丙酮酸。这再次伴随着增强的细菌溶血和溶血活性。总之，这些结果表明GGT在口腔细菌的谷胱甘肽代谢中起重要作用。
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 inTreponema denticolathat 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 fromT. denticola, which contains an open reading frame of 726 bp encoding a protein of 241 amino acids. Transformation of this gene intoEscherichia coliled 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 toT. 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 toPorphyromonas 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.