Larsson A, Sjöberg B M
EMBO J. 1986 Aug;5(8):2037-40. doi: 10.1002/j.1460-2075.1986.tb04461.x.
The small subunit of iron-dependent ribonucleotide reductases contains a stable organic free radical, which is essential for enzyme activity and which is localized to a tyrosine residue. Tyrosine-122 in the B2 subunit of Escherichia coli ribonucleotide reductase has been changed into a phenylalanine. The mutation was introduced with oligonucleotide-directed mutagenesis in an M13 recombinant and verified by DNA sequencing. Purified native and mutant B2 protein were found to have the same size, iron content and iron-related absorption spectrum. The sole difference observed is that the mutant protein lacks tyrosyl radical and enzymatic activity. These results identify Tyr122 of E. coli protein B2 as the tyrosyl radical residue. An expression vector was constructed for manipulation and expression of ribonucleotide reductase subunits. It contains the entire nrd operon with its own promoter in a 2.3-kb fragment from pBR322. Both the B1 and the B2 subunits were expressed at a 25-35 times higher level as compared to the host strain.
铁依赖性核糖核苷酸还原酶的小亚基含有一个稳定的有机自由基，该自由基对于酶活性至关重要且定位于一个酪氨酸残基上。大肠杆菌核糖核苷酸还原酶B2亚基中的酪氨酸-122已被替换为苯丙氨酸。该突变通过寡核苷酸定向诱变引入到一个M13重组体中，并经DNA测序验证。发现纯化的天然B2蛋白和突变型B2蛋白大小相同、铁含量相同且具有与铁相关的吸收光谱。观察到的唯一差异是突变蛋白缺乏酪氨酰自由基和酶活性。这些结果确定大肠杆菌蛋白B2的Tyr122为酪氨酰自由基残基。构建了一个用于操纵和表达核糖核苷酸还原酶亚基的表达载体。它在来自pBR322的一个2.3 kb片段中包含带有自身启动子的完整nrd操纵子。与宿主菌株相比，B1和B2亚基的表达水平均提高了25至35倍。
