Kannangara C G, Vothknecht U C, Hansson M, von Wettstein D
Department of Physiology, Carlsberg Laboratory, Copenhagen-Valby, Denmark.
Mol Gen Genet. 1997 Mar 18;254(1):85-92. doi: 10.1007/s004380050394.
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 Mg2+ 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 A260:A280 ratio of 1.8. On sucrose density gradients both Xantha-G and BchD subunits migrated with the plastid and bacterial ribosomal RNA, respectively.
镁螯合酶催化Mg2+插入原卟啉中，且仅在合成叶绿素或细菌叶绿素的生物体中发现。通过温和裂解大麦质体和球形红细菌球状体获得的可溶性蛋白质制剂，蛋白质含量>10 mg/ml，在ATP存在下，分别以40和8 pmoles/mg蛋白质每分钟的速率将Mg2+插入到二氢卟吩IX中。对于大麦提取物，在40 mM Mg2+时观察到最佳活性。该活性受到微摩尔浓度氯霉素的抑制。大麦中三个基因位点Xantha-f、-g和-h中的每一个发生突变都会破坏该活性。然而，通过将来自非渗漏型xantha-f、-g和-h突变体的质体基质蛋白质制剂两两组合，可在体外重建Mg螯合酶活性。这表明，与球形红细菌一样，在大麦中，将镁插入原卟啉IX需要三种蛋白质。这三种蛋白质，即Xantha-F、-G和-H，被称为Mg螯合酶亚基，它们在体内似乎彼此分离存在。将大麦和球形红细菌的活性制剂在272,000 x g下离心90分钟后，得到沉淀和上清部分。分别检测时，沉淀和上清均无活性，但将它们组合时活性得以恢复。通过使用来自xantha-f、-g和-h突变体的基质蛋白质进行体外互补分析，确定了Mg螯合酶亚基在各部分中的差异分布。Xantha-G蛋白局限于沉淀部分，而Xantha-H局限于上清部分。使用纯化的重组BchH、BchI和部分纯化的BchD进行重建分析表明，球形红细菌的沉淀部分含有BchD亚基。大麦和球形红细菌的沉淀部分均含有核糖体，A260:A280比值为1.8。在蔗糖密度梯度上，Xantha-G和BchD亚基分别与质体和细菌核糖体RNA一起迁移。
