Department of Agricultural, Food and Nutritional Science, University of Alberta, 4-10 Ag/For Centre, Edmonton, T6G 2P5, Canada.
School of Biology and Biological Engineering, South China University of Technology, Guangzhou, China.
Appl Microbiol Biotechnol. 2023 May;107(9):2997-3008. doi: 10.1007/s00253-023-12497-1. Epub 2023 Mar 30.
The γ-glutamyl tripeptide glutathione (γ-Glu-Cys-Gly) is a low molecular thiol that acts as antioxidant in response to oxidative stress in eukaryotes and prokaryotes. γ-Glutamyl dipeptides including γ-Glu-Cys, γ-Glu-Glu, and γ-Glu-Gly also have kokumi activity. Glutathione is synthesized by first ligating Glu with Cys by γ-glutamylcysteine ligase (Gcl/GshA), and then the resulting dipeptide γ-glutamylcysteine is ligated with Gly by glutathione synthetase (Gs/GshB). GshAB/GshF enzymes that contain both Gcl and Gs domains are capable of catalyzing both reactions. The current study aimed to characterize GshAB from Tetragenococcus halophilus after heterologous expression in Escherichia coli. The optimal conditions for GshAB from T. halophilus were pH 8.0 and 25 °C. The substrate specificity of the Gcl reaction of GshAB was also determined. GshAB has a high affinity to Cys. γ-Glu-Cys was the only dipeptide generated when Glu, Cys, Gly, and other amino acids were present in the reaction system. This specificity differentiates GshAB from T. halophilus from Gcl of heterofermentative lactobacilli and GshAB of Streptococcus agalactiae, which also use amino acids other than Cys as glutamyl-acceptor. Quantification of gshAB in cDNA libraries from T. halophilus revealed that gshAB was overexpressed in response to oxidative stress but not in response to acid, osmotic, or cold stress. In conclusion, GshAB in T. halophilus served as part of the oxidative stress response but this study did not provide any evidence for a contribution to the resistance to other stressors.Key points Glutathione synthesis in Tetragenococcus halophilus is carried out by the two-domain enzyme GshAB. GshAB is inhibited by glutathione and is highly specific for Cys as acceptor. T. halophilus synthesizes glutathione in response to oxidative stress.
γ-谷氨酰三肽谷胱甘肽（γ-Glu-Cys-Gly）是一种低分子量硫醇，可作为真核生物和原核生物氧化应激的抗氧化剂。γ-谷氨酰二肽，包括 γ-Glu-Cys、γ-Glu-Glu 和 γ-Glu-Gly，也具有 kokumi 活性。谷胱甘肽由 γ-谷氨酰半胱氨酸连接酶（Gcl/GshA）首先将 Glu 与 Cys 连接，然后将得到的二肽 γ-谷氨酰半胱氨酸与 Gly 由谷胱甘肽合成酶（Gs/GshB）连接。同时含有 Gcl 和 Gs 结构域的 GshAB/GshF 酶能够催化这两个反应。本研究旨在在大肠杆菌中异源表达后，对来自 Tetragenococcus halophilus 的 GshAB 进行表征。T. halophilus 的 GshAB 的最佳条件为 pH8.0 和 25°C。还确定了 Gcl 反应的底物特异性。GshAB 对 Cys 具有高亲和力。当 Glu、Cys、Gly 和其他氨基酸存在于反应体系中时，GshAB 仅产生 γ-Glu-Cys 二肽。这种特异性将 GshAB 与来自异型发酵乳杆菌的 Gcl 和来自乳链球菌的 GshAB 区分开来，后者也使用除 Cys 以外的氨基酸作为谷氨酰受体。T. halophilus cDNA 文库中 gshAB 的定量结果表明，gshAB 在氧化应激时表达上调，但在酸、渗透压和冷应激时不表达。总之，T. halophilus 中的 GshAB 作为氧化应激反应的一部分，但本研究没有提供任何证据表明其对其他应激源的抗性有贡献。关键点 Tetragenococcus halophilus 中的谷胱甘肽合成由双结构域酶 GshAB 进行。GshAB 受谷胱甘肽抑制，对 Cys 作为受体具有高度特异性。T. halophilus 在氧化应激时合成谷胱甘肽。
