Axarli Irene, Dhavala Prathusha, Papageorgiou Anastassios C, Labrou Nikolaos E
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, 75 Iera Odos Street, GR-11855-Athens, Greece.
J Mol Biol. 2009 Jan 23;385(3):984-1002. doi: 10.1016/j.jmb.2008.10.084. Epub 2008 Nov 6.
Glutathione transferases (GSTs) from the tau class (GSTU) are unique to plants and have important roles in stress tolerance and the detoxification of herbicides in crops and weeds. A fluorodifen-induced GST isoezyme (GmGSTU4-4) belonging to the tau class was purified from Glycine max by affinity chromatography. This isoenzyme was cloned and expressed in Escherichia coli, and its structural and catalytic properties were investigated. The structure of GmGSTU4-4 was determined at 1.75 A resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). The enzyme adopts the canonical GST fold but with a number of functionally important differences. Compared with other plant GSTs, the three-dimensional structure of GmGSTU4-4 primarily shows structural differences in the hydrophobic substrate binding site, the linker segment and the C-terminal region. The X-ray structure identifies key amino acid residues in the hydrophobic binding site (H-site) and provides insights into the substrate specificity and catalytic mechanism of the enzyme. The isoenzyme was highly active in conjugating the diphenylether herbicide fluorodifen. A possible reaction pathway involving the conjugation of glutathione with fluorodifen is described based on site-directed mutagenesis and molecular modeling studies. A serine residue (Ser13) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione and enhance its nucleophilicity. Tyr107 and Arg111 present in the active site are important structural moieties that modulate the catalytic efficiency and specificity of the enzyme, and participate in k(cat) regulation by affecting the rate-limiting step of the catalytic reaction. A hitherto undescribed ligand-binding site (L-site) located in a surface pocket of the enzyme was also found. This site is formed by conserved residues, suggesting it may have an important functional role in the transfer and delivery of bound ligands, presumably to specific protein receptors.
植物中的τ类谷胱甘肽转移酶（GSTU）是植物特有的，在作物和杂草的胁迫耐受性及除草剂解毒方面发挥着重要作用。通过亲和层析从大豆中纯化出一种属于τ类的氟乐灵诱导型GST同工酶（GmGSTU4-4）。对该同工酶进行了克隆并在大肠杆菌中表达，同时研究了其结构和催化特性。GmGSTU4-4与S-(对硝基苄基)-谷胱甘肽（Nb-GSH）形成复合物，其结构在1.75 Å分辨率下得以确定。该酶具有典型的GST折叠结构，但在功能上有一些重要差异。与其他植物GST相比，GmGSTU4-4的三维结构主要在疏水底物结合位点、连接片段和C端区域存在结构差异。X射线结构确定了疏水结合位点（H位点）中的关键氨基酸残基，并为该酶的底物特异性和催化机制提供了深入见解。该同工酶在偶联二苯醚除草剂氟乐灵方面具有高活性。基于定点诱变和分子模拟研究，描述了一个涉及谷胱甘肽与氟乐灵偶联的可能反应途径。活性位点存在一个丝氨酸残基（Ser13），其位置能够使其稳定谷胱甘肽的硫醇阴离子并增强其亲核性。活性位点中的Tyr107和Arg111是重要的结构部分，可调节酶的催化效率和特异性，并通过影响催化反应的限速步骤参与k(cat)调控。还发现了一个位于酶表面口袋中的此前未描述的配体结合位点（L位点）。该位点由保守残基形成，表明它可能在结合配体的转移和传递中发挥重要功能作用，推测是传递给特定的蛋白质受体。
