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Heterologous Expression of the Barley (Hordeum vulgare L.) Xantha-f, -g and -h Genes that Encode Magnesium Chelatase Subunits.
The Protein Journal ( IF 3 ) Pub Date : 2020-07-31 , DOI: 10.1007/s10930-020-09913-0
Rabab Mahdi 1 , David Stuart 1 , Mats Hansson 1 , Helmy M Youssef 1, 2
Affiliation  

Biosynthesis of chlorophyll involves several enzymatic reactions of which many are shared with the heme biosynthesis pathway. Magnesium chelatase is the first specific enzyme in the chlorophyll pathway. It catalyzes the formation of Mg-protoporphyrin IX from the insertion of Mg2+ into protoporphyrin IX. The enzyme consists of three subunits encoded by three genes. The three genes are named Xantha-h, Xantha-g and Xantha-f in barley (Hordeum vulgare L.). The products of the genes have a molecular weight of 38, 78 and 148 kDa, respectively, as mature proteins in the chloroplast. Most studies on magnesium chelatase enzymes have been performed using recombinant proteins of Rhodobacter capsulatus, Synechocystis sp. PCC6803 and Thermosynechococcus elongatus, which are photosynthetic bacteria. In the present study we established a recombinant expression system for barley magnesium chelatase with the long-term goal to obtain structural information of this enigmatic enzyme complex from a higher plant. The genes Xantha-h, -g and -f were cloned in plasmid pET15b, which allowed the production of the three subunits as His-tagged proteins in Escherichia coli BL21(DE3)pLysS. The purified subunits stimulated magnesium chelatase activity of barley plastid extracts and produced activity in assays with only recombinant proteins. In preparation for future structural analyses of the barley magnesium chelatase, stability tests were performed on the subunits and activity assays were screened to find an optimal buffer system and pH.



中文翻译:

编码镁螯合酶亚基的大麦(Hordeum vulgare L.)Xantha-f,-g和-h基因的异源表达。

叶绿素的生物合成涉及几种酶促反应,其中许多与血红素生物合成途径共有。镁螯合酶是叶绿素途径中的第一个特异性酶。它通过将Mg 2+插入原卟啉IX来催化Mg-原卟啉IX的形成。该酶由三个基因编码的三个亚基组成。三种基因被命名为黄化-H 黄化-G黄化-F在大麦(大麦L.)。这些基因的产物作为叶绿体中的成熟蛋白分别具有38、78和148 kDa的分子量。镁螯合酶的大多数研究已经使用荚膜红细菌的重组蛋白进行。集胞藻。PCC6803和嗜球菌是光合作用细菌。在本研究中,我们建立了大麦镁螯合酶的重组表达系统,其长期目标是从高等植物中获得这种神秘酶复合物的结构信息。将基因Xantha-h-g-f克隆到质粒pET15b中,从而可以在大肠杆菌中产生作为His标记蛋白的三个亚基。BL21(DE3)pLysS。纯化的亚基刺激了大麦质体提取物的镁螯合酶活性,并在仅使用重组蛋白的分析中产生了活性。在准备进行大麦镁螯合酶的未来结构分析时,对亚基进行了稳定性测试,并筛选了活性测定法以找到最佳的缓冲液系统和pH值。

更新日期:2020-07-31
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