Activation of the anaerobic ribonucleotide reductase from Escherichia coli - The essential role of the iron-sulfur center for S-adenosylmethionine reduction
Activation of the anaerobic ribonucleotide reductase from Escherichia coli - The essential role of the iron-sulfur center for S-adenosylmethionine reduction
复制标题
DOI：
10.1074/jbc.272.39.24216
复制
发表时间：
1997-09-26
影响因子：
4.8
通讯作者：
Fontecave, M
中科院分区：
文献类型：
--
作者：
Ollagnier, S;Mulliez, E;Fontecave, M
大肠杆菌的厌氧核糖核苷酸还原酶催化厌氧DNA合成所需的脱氧核糖核苷酸的合成。这种酶是一种α（2）β（2）异源四聚体。在其活性形式中，大的cu，亚基含有氧敏感性甘氨酰自由基，而β（2）小蛋白含有连接其两条多肽链的[4Fe-4S]簇。失活酶中甘氨酰自由基的形成需要S-腺苷甲硫氨酸（AdoMet）、二硫苏糖醇、K+以及酶（还原的黄氧还蛋白）或化学（连二亚硫酸盐或5-脱氮黄霉素加光）还原系统。在此，我们证明了在酶的活化过程中，β 2的Fe-S中心直接还原β 2的蛋氨酸，导致甲硫氨酸和甘氨酰自由基的形成。直接结合实验表明，β 2的蛋氨酸与β 2结合的Kd为10 μ M，化学计量比为1：1。EPR光谱证实了β 2的蛋氨酸与β 2的[4Fe-4S]中心之间形成复合物。二硫苏糖醇引发β-Met的切割，导致β（2）的EPR沉默形式，并且在α（2）β（2）的情况下，导致甘氨酰自由基的形成。在这两种情况下，每摩尔蛋白质形成3个甲硫氨酸。我们的结果表明，β（2）的Fe-S中心直接参与β-Met的还原切割，并暗示铁-硫中心的新生物学功能。即氧化还原催化，如最近由其他人提出的（斯台普斯，R，C，Ameyibor，E.，傅，W.，Gardet-Salvi，L，，Stritt-Etter，A. L，Schurmann，P，Knaff D，B，和约翰逊，M，K，（1996）生物化学35，11425-11434）。
The anaerobic ribonucleotide reductase of Escherichia coli catalyzes the synthesis of the deoxyribonucleotides required for anaerobic DNA synthesis. The enzyme is an alpha(2) beta(2) heterotetramer. in its active form, the large cu,subunit contains are oxygen-sensitive glycyl radical, whereas the beta(2) small protein harbors a [4Fe-4S] cluster that joins its two polypeptide chains, Formation of the glycyl radical in the inactive enzyme requires S-adenosylmethionine (AdoMet), dithiothreitol, K+, and either an enzymatic (reduced flavodoxin) or chemical (dithionite or 5-deazaflavin plus light) reducing system. Here, eve demonstrate that AdoMet is directly reduced by the Fe-S center of beta(2) during the activation of the enzyme, resulting in methionine and glycyl radical formation, Direct binding experiments showed that AdoMet binds to beta(2) with a K-d of 10 mu M and a 1:1 stoichiometry Binding was confirmed by EPR spectroscopy that demonstrated the formation of a complex between AdoMet and the [4Fe-4S] center of beta 2. Dithiothreitol triggered the cleavage of AdoMet, leading to an EPR-silent form of beta(2) and, in the case of alpha(2) beta(2), to glycyl radical formation, In both instances, 3 methionines were formed per mol of protein, Our results indicate that the Fe-S center of beta(2) is directly involved in the reductive cleavage of AdoMet and suggest a mew biological function for an iron-sulfur center, i.e redox catalysis, as recently proposed by others (Staples, R, C,, Ameyibor, E., Fu, W., Gardet-Salvi, L,, Stritt-Etter, A. L,, Schurmann, P,, Knaff D, B,, and Johnson, M, K, (1996) Biochemistry 35, 11425-11434).