Young P, Ohman M, Sjöberg B M
Department of Molecular Biology, Stockholm University, Sweden.
J Biol Chem. 1994 Nov 11;269(45):27815-8.
Bacteriophage T4 contains a phage-encoded anaerobic ribonucleoside triphosphate reductase, nrdD, previously named sunY. An open reading frame, 55.9, that resides downstream of the phage reductase was observed to have amino acid sequence similarity with the E. coli pyruvate formate-lyase (Pfl) activating enzyme. A stop codon was engineered into the cloned 55.9 gene and then recombined back into the phage genome. Phage-infected extracts that lack a functional 55.9 product have a 6-fold reduction in anaerobic ribonucleotide reductase activity and are unable to activate overexpressed T4 NrdD. Restoration of reductase activity was possible when 55.9- and nrdD- T4-infected Escherichia coli extracts were conjointly assayed. Comparing the anaerobic burst size of 55.9- infections to that of the parental phage indicates that anaerobic de novo synthesis of deoxyribonucleotides is nearly abolished in phage lacking the 55.9 product. We propose that T4 55.9 encodes an enzyme that activates T4 NrdD by generating a glycyl radical in the phage-encoded reductase. The homology between the Pfl activating enzyme and T4 55.9 product (in this communication renamed NrdG) in function as well as amino acid sequence is presumably a remnant of an ancient heritage between Pfl and the anaerobic ribonucleotide reductases.
噬菌体T4含有一种由噬菌体编码的厌氧核糖核苷三磷酸还原酶nrdD，该酶之前被命名为sunY。在噬菌体还原酶下游发现了一个开放阅读框55.9，其氨基酸序列与大肠杆菌丙酮酸甲酸裂解酶（Pfl）激活酶相似。在克隆的55.9基因中设计了一个终止密码子，然后将其重组回噬菌体基因组。缺乏功能性55.9产物的噬菌体感染提取物的厌氧核糖核苷酸还原酶活性降低了6倍，并且无法激活过表达的T4 NrdD。当联合检测55.9和nrdD感染的大肠杆菌提取物时，还原酶活性得以恢复。将55.9感染的噬菌体与亲本噬菌体的厌氧爆发量进行比较，结果表明，在缺乏55.9产物的噬菌体中，脱氧核糖核苷酸的厌氧从头合成几乎被消除。我们提出，T4 55.9编码一种酶，该酶通过在噬菌体编码的还原酶中产生甘氨酰自由基来激活T4 NrdD。Pfl激活酶与T4 55.9产物（在本通讯中重新命名为NrdG）在功能以及氨基酸序列上的同源性可能是Pfl与厌氧核糖核苷酸还原酶之间古老遗传关系的遗留。
