Pandey Tripti, Singh Sudhir Kumar, Chhetri Gaurav, Tripathi Timir, Singh Arvind Kumar
Molecular and Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong, India.
PLoS One. 2015 May 12;10(5):e0126811. doi: 10.1371/journal.pone.0126811. eCollection 2015.
Glutathione S-transferases (GSTs) are multifunctional enzymes present in virtually all organisms. Besides having an essential role in cellular detoxification, they also perform various other functions, including responses in stress conditions and signaling. GSTs are highly studied in plants and animals; however, the knowledge regarding GSTs in cyanobacteria seems rudimentary. In this study, we report the characterization of a highly pH stable GST from the model cyanobacterium--Synechocystis PCC 6803. The gene sll0067 was expressed in Escherichia coli (E. coli), and the protein was purified to homogeneity. The expressed protein exists as a homo-dimer, which is composed of about 20 kDa subunit. The results of the steady-state enzyme kinetics displayed protein's glutathione conjugation activity towards its class specific substrate- isothiocyanate, having the maximal activity with phenethyl isothiocyanate. Contrary to the poor catalytic activity and low specificity towards standard GST substrates such as 1-chloro-2,4-dinitrobenzene by bacterial GSTs, PmGST B1-1 from Proteus mirabilis, and E. coli GST, sll0067 has broad substrate degradation capability like most of the mammalian GST. Moreover, we have shown that cyanobacterial GST sll0067 is catalytically efficient compared to the best mammalian enzymes. The structural stability of GST was studied as a function of pH. The fluorescence and CD spectroscopy in combination with size exclusion chromatography showed a highly stable nature of the protein over a broad pH range from 2.0 to 11.0. To the best of our knowledge, this is the first GST with such a wide range of pH related structural stability. Furthermore, the presence of conserved Proline-53, structural motifs such as N-capping box and hydrophobic staple further aid in the stability and proper folding of cyanobacterial GST-sll0067.
谷胱甘肽S-转移酶（GSTs）是几乎存在于所有生物体中的多功能酶。除了在细胞解毒中发挥重要作用外，它们还执行各种其他功能，包括在应激条件下的反应和信号传导。GSTs在植物和动物中得到了深入研究；然而，关于蓝藻中GSTs的知识似乎还很基础。在本研究中，我们报道了来自模式蓝藻——聚球藻PCC 6803的一种高度pH稳定的GST的特性。基因sll0067在大肠杆菌（E. coli）中表达，并将蛋白质纯化至同质。表达的蛋白质以同源二聚体形式存在，由约20 kDa的亚基组成。稳态酶动力学结果显示该蛋白质对其类别特异性底物异硫氰酸酯具有谷胱甘肽结合活性，对苯乙基异硫氰酸酯具有最大活性。与细菌GSTs、奇异变形杆菌的PmGST B1-1和大肠杆菌GST对标准GST底物如1-氯-2,4-二硝基苯的催化活性差和特异性低相反，sll0067具有像大多数哺乳动物GST一样广泛的底物降解能力。此外，我们已经表明，与最好的哺乳动物酶相比，蓝藻GST sll0067具有高效催化性。研究了GST的结构稳定性与pH的关系。荧光和圆二色光谱结合尺寸排阻色谱显示，该蛋白质在2.0至11.0的宽pH范围内具有高度稳定的性质。据我们所知，这是第一个具有如此广泛pH相关结构稳定性的GST。此外，保守的脯氨酸-53、N-帽盒和疏水钉等结构基序的存在进一步有助于蓝藻GST-sll0067的稳定性和正确折叠。
