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首页> 外文期刊>Journal of bacteriology >Reconstitution of an Active Magnesium Chelatase Enzyme Complex from the bchI, -D, and-H Gene Products of the Green Sulfur BacteriumChlorobium vibrioforme Expressed inEscherichia coli
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Reconstitution of an Active Magnesium Chelatase Enzyme Complex from the bchI, -D, and-H Gene Products of the Green Sulfur BacteriumChlorobium vibrioforme Expressed inEscherichia coli

机译：从在大肠杆菌中表达的绿色硫细菌绿弧菌的bchI，-D和-H基因产物重建活性镁螯合酶复合物

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摘要

Magnesium-protoporphyrin chelatase, the first enzyme unique to the (bacterio)chlorophyll-specific branch of the porphyrin biosynthetic pathway, catalyzes the insertion of Mg2+ into protoporphyrin IX. Three genes, designated bchI,-D, and -H, from the strictly anaerobic and obligately phototrophic green sulfur bacterium Chlorobium vibrioforme show a significant level of homology to the magnesium chelatase-encoding genes bchI, -D, and-H and chlI, -D, and -Hof Rhodobacter sphaeroides and Synechocystisstrain PCC6803, respectively. These three genes were expressed inEscherichia coli; the subsequent purification of overproduced BchI and -H proteins on an Ni2+-agarose affinity column and denaturation of insoluble BchD protein in 6 M urea were required for reconstitution of Mg-chelatase activity in vitro. This work therefore establishes that the magnesium chelatase ofC. vibrioforme is similar to the magnesium chelatases of the distantly related bacteria R. sphaeroides andSynechocystis strain PCC6803 with respect to number of subunits and ATP requirement. In addition, reconstitution of an active heterologous magnesium chelatase enzyme complex was obtained by combining the C. vibrioforme BchI and -D proteins and theSynechocystis strain PCC6803 ChlH protein. Furthermore, two versions, with respect to the N-terminal start of the bchIgene product, were expressed in E. coli, yielding ca. 38- and ca. 42-kDa versions of the BchI protein, both of which proved to be active. Western blot analysis of these proteins indicated that two forms of BchI, corresponding to the 38- and the 42-kDa expressed proteins, are also present in C. vibrioforme.
机译：镁原卟啉螯合酶是卟啉生物合成途径的（细菌）叶绿素特异性分支所特有的第一个酶，它催化Mg 2 + 插入原卟啉IX中。严格厌氧和光养绿硫细菌 Chlorobium v​​ibrioforme bchI ， -D 和 -H 三个基因/ em>与镁螯合酶编码基因 bchI ， -D 和 -H 和 chlI具有显着的同源性， -D 和 -H 分别是球形芽孢杆菌（Rhodobacter sphaeroides）和球囊藻（Synechocystis）菌株PCC6803。这三个基因在大肠杆菌中表达。随后需要在Ni 2 + 琼脂糖亲和柱上纯化过量生成的BchI和-H蛋白，并在6 M尿素中使不溶性BchD蛋白变性，以在体外重建Mg-螯合酶活性。因此，这项工作确定了 C的镁螯合酶。弧菌类似于远缘细菌 R的镁螯合酶。球形芽孢杆菌和 Synechocystis 菌株PCC6803的亚基数量和ATP需求量。另外，通过结合 C获得活性异源镁螯合酶复合物的重构。弧菌 BchI和-D蛋白以及 Synechocystis 株PCC6803 ChlH蛋白。此外，关于 bchI 基因产物的N末端起始的两个版本在 E中表达。大肠杆菌，产生约38和BkI蛋白的42 kDa版本，都被证明具有活性。这些蛋白的蛋白质印迹分析表明， C中也存在两种形式的BchI，分别对应于38-和42-kDa表达的蛋白。弧菌。

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