Tauer A, Benner S A
Department of Chemistry, Eidgenössiche Technische Hochschule Zürich, Switzerland.
Proc Natl Acad Sci U S A. 1997 Jan 7;94(1):53-8. doi: 10.1073/pnas.94.1.53.
A coenzyme B12-dependent ribonucleotide reductase was purified from the archaebacterium Thermoplasma acidophila and partially sequenced. Using probes derived from the sequence, the corresponding gene was cloned, completely sequenced, and expressed in Escherichia coli. The deduced amino acid sequence shows that the catalytic domain of the B12-dependent enzyme from T. acidophila, some 400 amino acids, is related by common ancestry to the diferric tyrosine radical iron(III)-dependent ribonucleotide reductase from E. coli, yeast, mammalian viruses, and man. The critical cysteine residues in the catalytic domain that participate in the thiyl radical-dependent reaction have been conserved even though the cofactor that generates the radical is not. Evolutionary bridges created by the T. acidophila sequence and that of a B12-dependent reductase from Mycobacterium tuberculosis establish homology between the Fe-dependent enzymes and the catalytic domain of the Lactobacillus leichmannii B12-dependent enzyme as well. These bridges are confirmed by a predicted secondary structure for the Lactobacillus enzyme. Sequence similarities show that the N-terminal domain of the T. acidophila ribonucleotide reductase is also homologous to the anaerobic ribonucleotide reductase from E. coli, which uses neither B12 nor Fe cofactors. A predicted secondary structure of the N-terminal domain suggests that it is predominantly helical, as is the domain in the aerobic E. coli enzyme depending on Fe, extending the homologous family of proteins to include anaerobic ribonucleotide reductases, B12 ribonucleotide reductases, and Fe-dependent aerobic ribonucleotide reductases. A model for the evolution of the ribonucleotide reductase family is presented; in this model, the thiyl radical-based reaction mechanism is conserved, but the cofactor is chosen to best adapt the host organism to its environment. This analysis illustrates how secondary structure predictions can assist evolutionary analyses, each important in "post-genomic" biochemistry.
从嗜热栖热放线菌中纯化出一种依赖辅酶B12的核糖核苷酸还原酶，并对其进行了部分测序。利用从该序列推导得到的探针，克隆了相应基因，完成了全序列测定，并在大肠杆菌中进行了表达。推导的氨基酸序列表明，嗜热栖热放线菌中依赖B12的酶的催化结构域约400个氨基酸，与大肠杆菌、酵母、哺乳动物病毒和人类中依赖二价铁酪氨酸自由基铁(III)的核糖核苷酸还原酶具有共同的祖先关系。尽管产生自由基的辅因子不同，但参与基于硫自由基反应的催化结构域中的关键半胱氨酸残基得以保留。嗜热栖热放线菌序列与结核分枝杆菌中依赖B12的还原酶序列构建的进化桥梁，也确立了铁依赖酶与莱氏乳杆菌中依赖B12的酶的催化结构域之间的同源性。这些桥梁通过莱氏乳杆菌酶的预测二级结构得到证实。序列相似性表明，嗜热栖热放线菌核糖核苷酸还原酶的N端结构域也与大肠杆菌的厌氧核糖核苷酸还原酶同源，后者既不使用B12也不使用铁辅因子。N端结构域的预测二级结构表明，它主要是螺旋结构，与依赖铁的大肠杆菌有氧酶中的结构域一样，从而将同源蛋白质家族扩展到包括厌氧核糖核苷酸还原酶、B12核糖核苷酸还原酶和铁依赖的有氧核糖核苷酸还原酶。提出了一个核糖核苷酸还原酶家族的进化模型；在这个模型中，基于硫自由基的反应机制得以保留，但选择辅因子是为了使宿主生物体更好地适应其环境。这一分析说明了二级结构预测如何能够辅助进化分析，二者在“后基因组”生物化学中都很重要。
