Magnesium chelatase: Association with ribosomes and mutant complementation studies identify barley subunit Xantha-G as a functional counterpart of Rhodobacter subunit BchD
Magnesium chelatase: Association with ribosomes and mutant complementation studies identify barley subunit Xantha-G as a functional counterpart of Rhodobacter subunit BchD
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DOI：
10.1007/s004380050394
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发表时间：
1997-03-18
期刊：
影响因子：
--
通讯作者：
von Wettstein, D
中科院分区：
文献类型：
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作者：
Kannangara, CG;Vothknecht, UC;von Wettstein, D
镁螯合酶催化Mg 2+插入原卟啉，并且仅在合成叶绿素或细菌叶绿素的生物体中发现。可溶性蛋白质制剂，蛋白质含量>10 mg/ml，通过大麦质体和红细菌球质体的温和裂解获得。在ATP存在下，分别以每分钟40和8皮摩尔/毫克蛋白质的速率将Mg插入次卟啉IX中。对于大麦提取物，在40 mM Mg 2+下观察到最佳活性。微摩尔浓度的氯霉素抑制活性。在大麦中的三个遗传位点，Xantha-f，-g和-h中的每一个中的突变破坏了活性。然而，Mg-螯合酶活性在体外重建成对组合的质体基质蛋白制剂从非泄漏的黄嘌呤-f，-g和-h突变体。这表明，在Rhodobacter中，镁插入大麦中的原卟啉IX需要三种蛋白。这三种蛋白质Xantha-F、-G和-H被称为镁螯合酶亚基，它们在体内似乎彼此分开存在。在272000 x g下离心90 min后，来自大麦和红细菌的活性制剂产生沉淀和上清液级分。当分别测定时，沉淀和上清液是无活性的，但当它们组合时，活性恢复。通过使用来自xantha-f; -g和-h突变体的基质蛋白的体外互补测定，建立了组分中Mg-螯合酶亚基的差异分布。Xantha-G蛋白局限于沉淀部分，而Xantha-H局限于上清液。使用纯化的重组BchH，BchI和部分纯化的BchD的重建测定显示，来自红细菌的沉淀级分含有BchD亚基。来自大麦和红细菌的沉淀级分都含有核糖体，并且具有1.8的A（260）：A（280）比。在蔗糖密度梯度上，Xantha-G和BchD亚基分别随质体和细菌核糖体RNA迁移。
Magnesium chelatase catalyses the insertion of Mg2+ into protoporphyrin and is found exclusively in organisms which synthesise chlorophyll or bacteriochlorophyll. Soluble protein preparations containing >10 mg protein/ml, obtained by gentle lysis of barley plastids and Rhodobacter sphaeroplasts. inserted Mg into deuteroporphyrin IX in the presence of ATP at rates of 40 and 8 pmoles/mg protein per min, respectively. With barley extracts optimal activity was observed with 40 mM Mg2+. The activity was inhibited by micromolar concentrations of chloramphenicol. Mutations in each of three genetic loci, Xantha-f, -g and -h, in barley destroyed the activity. However, Mg-chelatase activity was reconstituted in vitro by combining pairwise the plastid stroma protein preparations from non-leaky xantha-f, -g and -h mutants. This establishes that, as in Rhodobacter three proteins are required for the insertion of magnesium into protoporphyrin IX in barley. These three proteins, Xantha-F, -G and -H, are referred to as Mg-chelatase subunits and they appear to exist separate from each other in vivo. Active preparations from barley and Rhodobacter yielded pellet and supernatant fractions upon centrifugation for 90 min at 272 000 x g. The pellet and the supernatant were inactive when assayed separately, but when they were combined activity was restored. Differential distribution of the Mg-chelatase subunits in the fractions was established by in vitro complementation assays using stroma protein From the xantha-f; -g, and -h mutants. Xantha-G protein was confined to the pellet fraction, while Xantha-H was confined to the supernatant. Reconstitution assays using purified recombinant BchH, BchI and partially purified BchD revealed that the pellet fraction from Rhodobacter contained the BchD subunit. The pellet fractions from both barley and Rhodobacter contained ribosomes and had an A(260):A(280) ratio of 1.8. On sucrose density gradients both Xantha-G and BchD subunits migrated with the plastid and bacterial ribosomal RNA, respectively.