Magnesium chelatase from Rhodobacter sphaeroides: initial characterization of the enzyme using purified subunits and evidence for a BchI-BchD complex
Magnesium chelatase from Rhodobacter sphaeroides: initial characterization of the enzyme using purified subunits and evidence for a BchI-BchD complex
复制标题
来自球形红杆菌的镁螯合酶：使用纯化亚基对该酶进行初步表征以及 BchI-BchD 复合物的证据
DOI：
10.1042/bj3370243
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发表时间：
1999
影响因子：
4.1
通讯作者：
C. Hunter
中科院分区：
文献类型：
--
作者：
L. Gibson;P. E. Jensen;C. Hunter
镁原卟啉IX螯合酶催化镁插入原卟啉IX，这是(细菌)叶绿素生物合成的第一步。在光合作用细菌球形红假单胞菌中，这个反应是由bchI、bchd和bchH基因的产物催化的。这些基因已经在大肠杆菌中表达，因此BchI、BchD和BchH蛋白都是通过N-末端His6亲和标签产生的，这使得通过一个层析步骤就可以产生大量高纯度、高活性的镁螯合酶亚基。此外，BCHD已经被净化，没有受到伴侣GroEL的污染，这在过去被证明是一个问题。BCHD在大肠杆菌中主要以不溶蛋白的形式存在，经6M尿素纯化后，加入BchI、MgCl2和ATP复性，得到高活性蛋白。用这种方法制备的BchI/BCHD混合物与BchH联合使用，测定了球形葡萄球菌镁螯合酶对其天然底物的动力学参数。我们首次证明了BchI和BCHD形成一个复合体，而建立和维持这个复合体需要镁离子和ATP。凝胶过滤数据表明，在镁离子和三磷酸腺苷存在下，BchI和BCHD形成了一个分子质量为200 kDa的复合体。我们的数据表明，在体内，BCHD只有在BchI、Mg2+和ATP存在的情况下才能正确折叠并保持正确的构象。
The enzyme magnesium-protoporphyrin IX chelatase (Mg chelatase) catalyses the insertion of Mg into protoporphyrin IX, the first committed step in (bacterio)chlorophyll biosynthesis. In the photosynthetic bacterium Rhodobacter sphaeroides, this reaction is catalysed by the products of the bchI, bchD and bchH genes. These genes have been expressed in Escherichia coli so that the BchI, BchD and BchH proteins are produced with N-terminal His6 affinity tags, which has led to the production of large amounts of highly purified, highly active Mg chelatase subunits from a single chromatography step. Furthermore, BchD has been purifed free of contamination with the chaperone GroEL, which had proven to be a problem in the past. BchD, present largely as an insoluble protein in E. coli, was purified in 6 M urea and refolded by addition of BchI, MgCl2 and ATP, yielding highly active protein. BchI/BchD mixtures prepared in this way were used in conjunction with BchH to determine the kinetic parameters of R. sphaeroides Mg chelatase for its natural substrates. We have been able to demonstrate for the first time that BchI and BchD form a complex, and that Mg2+ and ATP are required to establish and maintain this complex. Gel filtration data suggest that BchI and BchD form a complex of molecular mass 200 kDa in the presence of Mg2+ and ATP. Our data suggest that, in vivo, BchD is only folded correctly and maintained in its correct conformation in the presence of BchI, Mg2+ and ATP.