Pouliou Fotini, Perperopoulou Fereniki, Labrou Nikolaos E
Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, 75 Iera Odos Street, GR-11855-Athens. Greece.
Protein Pept Lett. 2017;24(10):922-935. doi: 10.2174/0929866524666171026125300.
Glutathione transferases (GSTs, EC. 2.5.1.18) form a large group of multifunctional enzymes that are involved in the metabolism and inactivation of a wide range of endogenous and xenobiotic compound as well as in cell regulation and response to several biotic and abiotic stresses.
In the present work, we report the comparative analysis of the structural and functional features of two isoenzymes (GmGSTU5-5 and GmGSTU8-8) of the glutathione transferase (GST) family from Glycine max.
Full-length cDNA clones of GmGSTU5-5 and GmGSTU8-8 were derived from RT-PCR of RNA isolated from soybean seedlings and were cloned into a T7 expression vector. Τhe recombinant enzymes were expressed in E. coli and purified by affinity chromatography. Substrate specificity, kinetic and inhibition analysis were carried out towards a range of different xenobiotic compounds and GSH analogues. The thermal stability of the enzymes was also evaluated using activity assays and differential scanning fluorimetry.
Analysis of substrate specificity using a range of thiol substrates and electrophilic compounds suggested that both isoenzymes display broad and overlapping specificities. They are capable of detoxifying major stress-induced toxic products. Study of their ligandin-binding properties by kinetic analysis and molecular modelling indicated that both GmGSTU5-5 and GmGSTU8-8 bind a range of secondary metabolites and plant hormones, suggesting a role in transport or storage of bioactive compounds. Thermostability analysis showed that GmGSTU5-5 and GmGSTU8-8 display extraordinary thermal stability, compared to other plant GSTs.
Our results suggest that GmGSTU5-5 and GmGSTU8-8 display different or overlapping substrate specificities and kinetic properties. The biological role of GmGSTU5-5 and GmGSTU8-8 may be relevant to the detoxification of toxic compounds or the binding of bioactive metabolites that function in cell regulation and stress defence mechanisms.
谷胱甘肽转移酶（GSTs，EC. 2.5.1.18）构成了一大类多功能酶，它们参与多种内源性和外源性化合物的代谢与失活，以及细胞调节和对多种生物和非生物胁迫的反应。
在本研究中，我们报告了来自大豆的谷胱甘肽转移酶（GST）家族的两种同工酶（GmGSTU5-5和GmGSTU8-8）的结构和功能特征的比较分析。
GmGSTU5-5和GmGSTU8-8的全长cDNA克隆来自从大豆幼苗中分离的RNA的逆转录聚合酶链反应（RT-PCR），并被克隆到T7表达载体中。重组酶在大肠杆菌中表达并通过亲和层析纯化。针对一系列不同的外源性化合物和谷胱甘肽（GSH）类似物进行底物特异性、动力学和抑制分析。还使用活性测定和差示扫描荧光法评估了酶的热稳定性。
使用一系列硫醇底物和亲电子化合物进行的底物特异性分析表明，两种同工酶都表现出广泛且重叠的特异性。它们能够解毒主要的胁迫诱导毒性产物。通过动力学分析和分子建模对它们的配体结合特性的研究表明，GmGSTU5-5和GmGSTU8-8都结合一系列次生代谢物和植物激素，表明它们在生物活性化合物的运输或储存中起作用。热稳定性分析表明，与其他植物GST相比，GmGSTU5-5和GmGSTU8-8表现出非凡的热稳定性。
我们的结果表明，GmGSTU5-5和GmGSTU8-8表现出不同或重叠的底物特异性和动力学特性。GmGSTU5-5和GmGSTU8-8的生物学作用可能与有毒化合物的解毒或在细胞调节和胁迫防御机制中起作用的生物活性代谢物的结合有关。
