Department of Molecular Enzymology, Institute of Biochemistry and Biology, University of Potsdam, 14476 Potsdam, Germany.
J Biol Chem. 2011 Jan 14;286(2):1400-8. doi: 10.1074/jbc.M110.155671. Epub 2010 Nov 16.
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.
钼辅因子通过在钼喋呤的 C4' 磷酸上加 GMP 或 CMP 分别形成鸟嘌呤二核苷酸或胞嘧啶二核苷酸辅因子而被修饰。这两个反应分别由特定的酶催化：GTp：钼喋呤鸟苷酰转移酶 MobA 和 CTP：钼喋呤胞苷酰转移酶 MocA。两种酶的氨基酸序列同一性为 22%，并且对其各自的核苷酸具有特异性。MobA 的晶体结构分析揭示了蛋白质 N 端结构域中两个与鸟嘌呤碱基结合相关的保守基序。基于这些基序，我们进行了定点突变研究，将氨基酸交换为在同工酶 MocA 中发现的序列。使用完全定义的体外系统，我们表明，在 MocA 或 MobA 中，交换五个氨基酸足以获得与 GTP 和 CTP 的活性。每个蛋白质的完整 N 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 端结构域决定核苷酸的识别和结合。蛋白质-蛋白质相互作用的分析表明，MocA 或 MobA 的 C 端结构域决定了与各自的受体蛋白的特异性结合。
