Molybdopterin Dinucleotide Biosynthesis in Escherichia coli IDENTIFICATION OF AMINO ACID RESIDUES OF MOLYBDOPTERIN DINUCLEOTIDE TRANSFERASES THAT DETERMINE SPECIFICITY FOR BINDING OF GUANINE OR CYTOSINE NUCLEOTIDES
Molybdopterin Dinucleotide Biosynthesis in Escherichia coli IDENTIFICATION OF AMINO ACID RESIDUES OF MOLYBDOPTERIN DINUCLEOTIDE TRANSFERASES THAT DETERMINE SPECIFICITY FOR BINDING OF GUANINE OR CYTOSINE NUCLEOTIDES
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DOI：
10.1074/jbc.m110.155671
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发表时间：
2011-01-14
影响因子：
4.8
通讯作者：
Leimkuehler, Silke
中科院分区：
文献类型：
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作者：
Neumann, Meina;Seduk, Farida;Leimkuehler, Silke
通过将GMP或CMP分别添加到钼杂喋呤的C4‘磷酸中，形成钼杂蝶呤鸟嘌呤二核苷酸或胞嘧啶二核苷酸辅因子来修饰钼辅因子。这两个反应是由特定的酶催化的，它们分别是：GTP：钼杂蛋白鸟苷转移酶MOBA和CTP：钼杂蛋白胞苷转移酶MOCA。这两种酶都显示出22%的氨基酸序列同源性，并对各自的核苷酸具有特异性。对MOBA的晶体结构分析表明，在参与鸟嘌呤碱基结合的蛋白质的N-末端结构域上有两个保守的基序。基于这些基序，我们进行了定点突变研究，将氨基酸交换到准分子MOCA中的序列。使用完全定义的体外系统，我们证明了在MOCA或MOBA中，五个氨基酸的交换足以与GTP和CTP获得活性。每个蛋白质的完整N-末端结构域的交换导致核苷酸特异性活性的完全反转，表明N-末端结构域决定核苷酸的识别和结合。对蛋白质相互作用的分析表明，MOCA或MOBA的C-末端结构域决定了与各自受体蛋白的特异性结合。
The molybdenum cofactor is modified by the addition of GMP or CMP to the C4' phosphate of molybdopterin forming the molybdopterin guanine dinucleotide or molybdopterin cytosine dinucleotide cofactor, respectively. The two reactions are catalyzed by specific enzymes as follows: the GTP: molybdopterin guanylyltransferase MobA and the CTP: molybdopterin cytidylyltransferase MocA. Both enzymes show 22% amino acid sequence identity and are specific for their respective nucleotides. Crystal structure analysis of MobA revealed two conserved motifs in the N-terminal domain of the protein involved in binding of the guanine base. Based on these motifs, we performed site-directed mutagenesis studies to exchange the amino acids to the sequence found in the paralogue MocA. Using a fully defined in vitro system, we showed that the exchange of five amino acids was enough to obtain activity with both GTP and CTP in either MocA or MobA. Exchange of the complete N-terminal domain of each protein resulted in the total inversion of nucleotide specificity activity, showing that the N-terminal domain determines nucleotide recognition and binding. Analysis of protein-protein interactions showed that the C-terminal domain of either MocA or MobA determines the specific binding to the respective acceptor protein.