Gromes Roland, Hothorn Michael, Lenherr Esther D, Rybin Vladimir, Scheffzek Klaus, Rausch Thomas
Heidelberg Institute for Plant Sciences, Molecular Ecophysiology, Im Neuenheimer Feld 360, 69120 Heidelberg, Germany.
Plant J. 2008 Jun;54(6):1063-75. doi: 10.1111/j.1365-313X.2008.03477.x. Epub 2008 Mar 12.
In plants, the first committed enzyme for glutathione biosynthesis, gamma-glutamylcysteine ligase (GCL), is under multiple controls. The recent elucidation of GCL structure from Brassica juncea (BjGCL) has revealed the presence of two intramolecular disulfide bridges (CC1, CC2), which both strongly impact on GCL activity in vitro. Here we demonstrate that cysteines of CC1 are confined to plant species from the Rosids clade, and are absent in other plant families. Conversely, cysteines of CC2 involved in the monomer-dimer transition in BjGCL are not only conserved in the plant kingdom, but are also conserved in the evolutionarily related alpha- (and some gamma-) proteobacterial GCLs. Focusing on the role of CC2 for GCL redox regulation, we have extended our analysis to all available plant (31; including moss and algal) and related proteobacterial GCL (46) protein sequences. Amino acids contributing to the homodimer interface in BjGCL are highly conserved among plant GCLs, but are not conserved in related proteobacterial GCLs. To probe the significance of this distinction, recombinant GCLs from Nicotiana tabacum (NtGCL), Agrobacterium tumefaciens (AtuGCL, alpha-proteobacteria) and Xanthomonas campestris (XcaGCL, gamma-proteobacteria) were analyzed for their redox response. As expected, NtGCL forms a homodimer under oxidizing conditions, and is activated more than threefold. Conversely, proteobacterial GCLs remain monomeric under oxidizing and reducing conditions, and their activities are not inhibited by DTT, despite the presence of CC2. We conclude that although plant GCLs are evolutionarily related to proteobacterial GCLs, redox regulation of their GCLs via CC2-dependent dimerization has been acquired later in evolution, possibly as a consequence of compartmentation in the redox-modulated plastid environment.
在植物中，谷胱甘肽生物合成的首个关键酶γ-谷氨酰半胱氨酸连接酶（GCL）受到多种调控。最近对芥菜（BjGCL）的GCL结构解析发现，其存在两个分子内二硫键（CC1、CC2），这两个二硫键均对体外GCL活性有强烈影响。在此，我们证明CC1的半胱氨酸仅限于蔷薇分支的植物物种，在其他植物科中不存在。相反，参与BjGCL单体-二聚体转变的CC2半胱氨酸不仅在植物界保守，在进化相关的α-（以及一些γ-）变形菌GCL中也保守。聚焦于CC2对GCL氧化还原调节的作用，我们将分析扩展到所有可用的植物（31种；包括苔藓和藻类）及相关变形菌GCL（46种）蛋白质序列。在植物GCL中，构成BjGCL同型二聚体界面的氨基酸高度保守，但在相关变形菌GCL中不保守。为探究这种差异的意义，对烟草（NtGCL）、根癌农杆菌（AtuGCL，α-变形菌）和野油菜黄单胞菌（XcaGCL，γ-变形菌）的重组GCL进行了氧化还原反应分析。如预期的那样，NtGCL在氧化条件下形成同型二聚体，并被激活三倍以上。相反，变形菌GCL在氧化和还原条件下均保持单体状态，尽管存在CC2，其活性也不受二硫苏糖醇（DTT）抑制。我们得出结论，尽管植物GCL在进化上与变形菌GCL相关，但通过CC2依赖性二聚化对其GCL进行氧化还原调节是在进化后期获得的，这可能是氧化还原调节的质体环境中分隔的结果。
