Cloning and expression of the tobacco CHLM sequence encoding Mg protoporphyrin IX methyltransferase and its interaction with Mg chelatase
Cloning and expression of the tobacco CHLM sequence encoding Mg protoporphyrin IX methyltransferase and its interaction with Mg chelatase
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DOI：
10.1007/s11103-005-1427-8
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发表时间：
2005-03-01
影响因子：
5.1
通讯作者：
Grimm, B
中科院分区：
文献类型：
--
作者：
Alawady, A;Reski, R;Grimm, B
S-腺苷-L-甲硫氨酸：镁-原卟啉IX甲基转移酶（MgPMT）是四吡咯生物合成途径的镁分支中的一种酶。鉴定了烟草（Nicotiana tabacum）CHLM的核苷酸序列，并将cDNA序列用于表达前体、成熟的和截短的重组MgPMT，用于酶活性测试和多克隆抗体的形成。比较成熟的和截短的MgPMT揭示了三个关键的氨基酸在N-末端的MgPMT酶活性的维持。为了评估CHLM表达对控制四吡咯途径中代谢流的贡献，在幼苗绿化和植物发育期间以及在日/夜和连续生长条件下分析了CHLM转录物和蛋白质水平、酶活性和Mg原卟啉和Mg原卟啉单甲酯的稳态水平。这些表达研究揭示了MgPMT在绿化和光/暗周期中的翻译后激活。利用酵母双杂交系统的物理相互作用之间的MgPMT和镁螯合酶的CHLH亚基证明。在酵母细胞中表达的重组MgPMT的活性在重组CHLH亚基的存在下被刺激。翻译后调节MgPMT的影响进行了讨论的镁分支的开始酶的步骤。
S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase (MgPMT) is an enzyme in the Mg branch of the tetrapyrrole biosynthetic pathway. The nucleotide sequence of tobacco (Nicotiana tabacum) CHLM was identified and the cDNA sequence was used to express the precursor, the mature and a truncated recombinant MgPMT for enzymatic activity tests and for the formation of polyclonal antibodies. Comparison of the mature and the truncated MgPMT revealed three critical amino acids at the N-terminus of MgPMT for the maintenance of enzyme activity. To assess the contribution of CHLM expression to the control of the metabolic flow in the tetrapyrrole pathway, CHLM transcripts and protein levels, the enzyme activity and the steady-state levels of Mg protoporphyrin and Mg protoporphyrin monomethylester were analysed during greening of seedlings and plant development as well as under day/night and continuous growth conditions. These expression studies revealed posttranslational activation of MgPMT during greening and light/dark-cycles. Using the yeast two-hybrid system physical interaction was demonstrated between MgPMT and the CHLH subunit of Mg chelatase. Activity of recombinant MgPMT expressed in yeast cells was stimulated in the presence of the recombinant CHLH subunit. Implications for posttranslational regulation of MgPMT are discussed for the enzymatic steps at the beginning of the Mg branch.