Howell M L, Sanders-Loehr J, Loehr T M, Roseman N A, Mathews C K, Slabaugh M B
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-6503.
J Biol Chem. 1992 Jan 25;267(3):1705-11.
During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.
在其感染周期中，痘苗病毒表达一种病毒编码的核糖核苷酸还原酶，该酶与宿主细胞酶不同（斯拉博，M.B.，和马修斯，C.K.（1984年）《病毒学杂志》52卷，501 - 506页；斯拉博，M.B.，约翰逊，T.L.，和马修斯，C.K.（1984年）《病毒学杂志》52卷，507 - 514页）。我们已将痘苗病毒核糖核苷酸还原酶小亚基（命名为VVR2）的基因克隆到大肠杆菌中，并使用T7 RNA聚合酶质粒表达系统表达该蛋白。经异丙基β - D - 硫代半乳糖苷诱导后，通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳检测到一种37 kDa肽的积累，并且该肽与针对TrpE - VVR2融合蛋白产生的多克隆抗血清发生反应。将37 kDa蛋白纯化至同质，对纯化蛋白进行凝胶过滤显示重组蛋白在溶液中以二聚体形式存在。已证明纯化的重组VVR2蛋白可补充纯化的重组核糖核苷酸还原酶大亚基的活性，其比活性与来自病毒感染细胞提取物的天然VVR2相似。重组病毒蛋白的圆二色光谱表明，与小鼠蛋白一样，痘苗病毒蛋白具有50%的α - 螺旋结构。与其他含铁核糖核苷酸还原酶小亚基一样，重组VVR2蛋白含有一个稳定的有机自由基，可通过电子顺磁共振光谱检测到。纯化的重组VVR2的电子顺磁共振光谱与痘苗病毒感染的哺乳动物细胞的光谱相同。VVR2的超精细分裂特征和微波饱和行为均与小鼠R2相似，且与大肠杆菌R2不同。通过氨基酸分析确定VVR2的浓度，我们测定每个R2二聚体中约存在0.6个自由基。我们的结果表明痘苗病毒小亚基与哺乳动物核糖核苷酸还原酶相似。
