Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, 75 Iera Odos Street, 11855, Athens, Greece.
Appl Biochem Biotechnol. 2014 Jan;172(2):595-609. doi: 10.1007/s12010-013-0509-3. Epub 2013 Oct 9.
Glutathione transferases (GSTs, EC 2.5.1.18) are ubiquitous proteins in plants that play important roles in stress tolerance and in the detoxification of toxic chemicals and metabolites. In this study, we systematically examined the catalytic diversification of a GST isoenzyme from Phaseolus vulgaris (PvGST) which is induced under biotic stress treatment (Uromyces appendiculatus infection). The full-length cDNA of this GST isoenzyme (termed PvGSTU3-3) with complete open reading frame, was isolated using RACE-RT and showed that the deduced amino acid sequence shares high homology with the tau class plant GSTs. PvGSTU3-3 catalyzes several different reactions and exhibits wide substrate specificity. Of particular importance is the finding that the enzyme shows high antioxidant catalytic function and acts as hydroperoxidase, thioltransferase, and dehydroascorbate reductase. In addition, its K m for GSH is about five to ten times lower compared to other plant GSTs, suggesting that PvGSTU3-3 is able to perform efficient catalysis under conditions where the concentration of reduced glutathione is low (e.g., oxidative stress). Its ability to conjugate GSH with isothiocyanates may provide an additional role for this enzyme to act as a regulator of the released isothiocyanates from glucosinolates as a response of biotic stress. Molecular modeling showed that PvGSTU3-3 shares the same overall fold and structural organization with other plant cytosolic GSTs, with major differences at their hydrophobic binding sites (H-sites) and some differences at the level of C-terminal domain and the linker between the C- and N-terminal domains. PvGSTU3-3, in general, exhibits restricted ability to bind xenobiotics in a nonsubstrate manner, suggesting that the biological role of PvGSTU3-3, is restricted mainly to the catalytic function. Our findings highlight the functional and catalytic diversity of plant GSTs and demonstrate their pivotal role for addressing biotic stresses in Phaseolus vulgaris.
谷胱甘肽转移酶（GSTs，EC 2.5.1.18）是植物中普遍存在的蛋白质，在应激耐受和有毒化学物质和代谢物的解毒中发挥重要作用。在这项研究中，我们系统地研究了一种来自菜豆（Phaseolus vulgaris）的 GST 同工酶（称为 PvGSTU3-3）的催化多样性，该同工酶在生物胁迫处理（Uromyces appendiculatus 感染）下被诱导。使用 RACE-RT 分离了该 GST 同工酶的全长 cDNA，具有完整的开放阅读框，并且推断的氨基酸序列与 tau 类植物 GSTs 具有高度同源性。PvGSTU3-3 催化几种不同的反应，并表现出广泛的底物特异性。特别重要的是发现该酶具有高抗氧化催化功能，并作为过氧化物酶、硫转移酶和脱氢抗坏血酸还原酶发挥作用。此外，其对 GSH 的 K m 比其他植物 GST 低约五到十倍，这表明 PvGSTU3-3 能够在还原型谷胱甘肽浓度低（例如，氧化应激）的条件下进行有效的催化。其与异硫氰酸酯结合 GSH 的能力可能为该酶提供了另一个作用，作为生物胁迫响应中从硫代葡萄糖苷释放的异硫氰酸酯的调节剂。分子建模表明，PvGSTU3-3 与其他植物细胞质 GST 具有相同的整体折叠和结构组织，在其疏水性结合位点（H 位点）和 C 末端结构域和 C-和 N 末端结构域之间的连接体的某些水平上存在较大差异。PvGSTU3-3 通常表现出以非底物方式结合外源性物质的能力有限，这表明 PvGSTU3-3 的生物学作用主要限于催化功能。我们的发现突出了植物 GSTs 的功能和催化多样性，并证明了它们在应对菜豆生物胁迫方面的关键作用。
