Willing A, Follmann H, Auling G
Fachbereich Chemie der Philipps-Universität Marburg, Federal Republic of Germany.
Eur J Biochem. 1988 Jan 4;170(3):603-11. doi: 10.1111/j.1432-1033.1988.tb13740.x.
Ribonucleotide reduction and not DNA replication is the site for the specific manganese requirement of DNA synthesis and cell growth in the coryneform bacterium Brevibacterium ammoniagenes. To characterize the metal effect we have isolated and purified ribonucleoside-diphosphate reductase from overproducing bacteria that were first deprived of and then reactivated by manganese ions. Purification on columns of Sephacryl S400, DEAE-cellulose and hydroxyapatite provided an apparently homogeneous enzyme consisting of two protein subunits. These were characterized by affinity chromatography on 2',5'-ADP-Sepharose as nucleotide-binding protein B1 (Mr = 80,000) and catalytic protein B2 (Mr = 100,000, composed of two Mr = 50,000 polypeptides), which were both necessary for activity. In vitro the purified enzyme does not require added metal ions except for an unspecific, twofold activity increase observed in the presence of Mg2+ and other divalent cations. Enzyme activity is inhibited by hydroxyurea (I50 = 2.5 mM). The electronic spectrum with maxima around 455 nm and 485 nm closely resembles that of manganese(III)-containing pseudocatalase and of oxo-bridged binuclear Mn(III) model complexes. Denaturation of the enzyme in trichloroacetic acid liberated an equimolar amount of Mn(II) which was detected by EPR spectroscopy. It was not possible to remove and reintroduce metal ions without loss of enzyme activity. Manganese-deficient cell cultures were also grown in the presence of 54MnCl2. Ribonucleotide reductase activity and radioactivity cochromatographed in several systems. Non-denaturing polyacrylamide gel electrophoresis showed that protein subunit B2 was specifically 54Mn-labeled. All these properties suggest that the ribonucleotide reductase of B. ammoniagenes is a manganese-containing analog of the non-heme-iron-containing reductases of Escherichia coli and eukaryotes.
在棒状杆菌属的产氨短杆菌中，核糖核苷酸还原而非DNA复制是DNA合成和细胞生长对特定锰需求的位点。为了表征金属效应，我们从过量生产的细菌中分离并纯化了核糖核苷二磷酸还原酶，这些细菌首先被剥夺锰离子，然后再用锰离子重新激活。通过Sephacryl S400、DEAE - 纤维素和羟基磷灰石柱进行纯化，得到了一种明显均一的酶，该酶由两个蛋白质亚基组成。通过在2',5'-ADP - 琼脂糖上进行亲和色谱分析，这些亚基被表征为核苷酸结合蛋白B1（Mr = 80,000）和催化蛋白B2（Mr = 100,000，由两个Mr = 50,000的多肽组成），二者对于活性都是必需的。在体外，纯化后的酶除了在Mg2+和其他二价阳离子存在下观察到非特异性的活性增加两倍外，不需要添加金属离子。酶活性受到羟基脲的抑制（I50 = 2.5 mM）。最大吸收峰在455 nm和485 nm附近的电子光谱与含锰(III)的假过氧化氢酶以及氧桥联双核锰(III)模型配合物的电子光谱非常相似。在三氯乙酸中使酶变性会释放出等摩尔量的Mn(II)，通过电子顺磁共振光谱检测到了该离子。在不损失酶活性的情况下，去除和重新引入金属离子是不可能的。缺锰的细胞培养物也在54MnCl2存在下生长。核糖核苷酸还原酶活性和放射性在几个系统中共同色谱分离。非变性聚丙烯酰胺凝胶电泳表明蛋白质亚基B2被特异性地54Mn标记。所有这些特性表明，产氨短杆菌的核糖核苷酸还原酶是大肠杆菌和真核生物中含非血红素铁还原酶的含锰类似物。
