Structural and functional consequences of removing the N-terminal domain from the magnesium chelatase ChlH subunit of Thermosynechococcus elongatus.
Structural and functional consequences of removing the N-terminal domain from the magnesium chelatase ChlH subunit of Thermosynechococcus elongatus.
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DOI：
10.1042/bj20140463
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发表时间：
2014-12-15
期刊：
影响因子：
--
通讯作者：
Hunter CN
中科院分区：
文献类型：
--
作者：
Adams NB;Marklew CJ;Qian P;Brindley AA;Davison PA;Bullough PA;Hunter CN
镁螯合酶（Magnesium chelatase, MgCH）通过催化atp依赖的Mg2+插入到原卟啉中来启动叶绿素的生物合成。这种大型酶复合体包括ChlH、I和D亚基，其中I和D参与ATP水解，H是处理底物和产物的蛋白质。148 kDa ChlH亚基有一个球状n端结构域，通过一个狭窄的连接体连接到一个中空的笼状结构上。在长形热共胞菌ChlH中缺失这一~18 kDa结构域后，我们使用单粒子重建表明，突变亚基的载子和卟啉结合形式由一个中空的球状蛋白组成，具有三个相连的裂片；突变体和天然ChlH结构的叠加表明，尽管n端“头部”区域明显缺失，但蛋白质的其余部分似乎是正确折叠的。解离常数分析表明，ΔN159ChlH突变体保留了结合原卟啉和Gun4增强蛋白的能力，尽管添加I和D亚基会产生严重受损的活性酶复合物。Gun4增强子蛋白的加入可以显著刺激MgCH活性，特别是在低Mg2+浓度下，部分重新激活ΔN159ChlH-I-D突变酶复合物，这表明Gun4在H上的结合位点并不完全依赖于n端结构域。148 kDa ChlH的n端结构域对ChlH - i - d镁螯合酶复合物的正常活性至关重要。删除这个18kda结构域保留了中空的笼状结构和卟啉结合。Gun4蛋白部分恢复螯合酶活性。
Magnesium chelatase (MgCH) initiates chlorophyll biosynthesis by catalysing the ATP-dependent insertion of Mg2+ into protoporphyrin. This large enzyme complex comprises ChlH, I and D subunits, with I and D involved in ATP hydrolysis, and H the protein that handles the substrate and product. The 148 kDa ChlH subunit has a globular N-terminal domain attached by a narrow linker to a hollow cage-like structure. Following deletion of this ~18 kDa domain from the Thermosynechoccus elongatus ChlH, we used single particle reconstruction to show that the apo- and porphyrin-bound forms of the mutant subunit consist of a hollow globular protein with three connected lobes; superposition of the mutant and native ChlH structures shows that, despite the clear absence of the N-terminal ‘head’ region, the rest of the protein appears to be correctly folded. Analyses of dissociation constants shows that the ΔN159ChlH mutant retains the ability to bind protoporphyrin and the Gun4 enhancer protein, although the addition of I and D subunits yields an extremely impaired active enzyme complex. Addition of the Gun4 enhancer protein, which stimulates MgCH activity significantly especially at low Mg2+ concentrations, partially reactivates the ΔN159ChlH–I–D mutant enzyme complex, suggesting that the binding site or sites for Gun4 on H do not wholly depend on the N-terminal domain. The N-terminal domain of the 148 kDa ChlH is essential for normal activity of the ChlH–I–D magnesium chelatase complex. Deleting this 18 kDa domain retains the hollow cage-like structure and porphyrin binding. Chelatase activity is partially restored by the Gun4 protein.