Roshick C, Iliffe-Lee E R, McClarty G
Department Of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba R3E 0W3, Canada.
J Biol Chem. 2000 Dec 1;275(48):38111-9. doi: 10.1074/jbc.M006367200.
In all organisms the deoxyribonucleotide precursors required for DNA synthesis are synthesized from ribonucleotides, a reaction catalyzed by ribonucleotide reductase. In a previous study we showed that Chlamydia trachomatis growth was inhibited by hydroxyurea, an inhibitor of ribonucleotide reductase, and a mutant resistant to the cytotoxic effects of the drug was isolated. Here we report the cloning, expression, and purification of the R1 and R2 subunits of the C. trachomatis ribonucleotide reductase. In comparison with other ribonucleotide reductases, the primary sequence of protein R1 has an extended amino terminus, and the R2 protein has a phenylalanine where the essential tyrosine is normally located. Despite its unusual primary structure, the recombinant enzyme catalyzes the reduction of CDP to dCDP. Results from deletion mutagenesis experiments indicate that while the extended amino terminus of the R1 protein is not required for enzyme activity, it is needed for allosteric inhibition mediated by dATP. Results with site-directed mutants of protein R2 suggest that the essential tyrosine is situated two amino acids downstream of its normal location. Finally, Western blot analysis show that the hydroxyurea-resistant mutant C. trachomatis isolate overexpresses both subunits of ribonucleotide reductase. At the genetic level, compared with wild type C. trachomatis, the resistant isolate has a single base mutation just upstream of the ATG start codon of the R2 protein. The possibility that this mutation affects translational efficiency is discussed.
在所有生物体中，DNA合成所需的脱氧核糖核苷酸前体都是由核糖核苷酸合成的，这一反应由核糖核苷酸还原酶催化。在之前的一项研究中，我们发现沙眼衣原体的生长受到核糖核苷酸还原酶抑制剂羟基脲的抑制，并且分离出了对该药物细胞毒性作用具有抗性的突变体。在此，我们报告沙眼衣原体核糖核苷酸还原酶R1和R2亚基的克隆、表达及纯化。与其他核糖核苷酸还原酶相比，蛋白质R1的一级序列具有延长的氨基末端，而R2蛋白在通常为必需酪氨酸的位置上是苯丙氨酸。尽管其一级结构异常，但重组酶仍能催化CDP还原为dCDP。缺失诱变实验结果表明，虽然R1蛋白延长的氨基末端对于酶活性不是必需的，但它是dATP介导的变构抑制所必需的。蛋白质R2定点突变体的结果表明，必需酪氨酸位于其正常位置下游两个氨基酸处。最后，蛋白质印迹分析表明，对羟基脲具有抗性的沙眼衣原体突变体分离株中核糖核苷酸还原酶的两个亚基均过表达。在基因水平上，与野生型沙眼衣原体相比，抗性分离株在R2蛋白的ATG起始密码子上游有一个单碱基突变。文中讨论了该突变影响翻译效率的可能性。
