Efficient carbon dioxide fixation by a Rubisco from hyperthermophilic archaea

我们的特点是基因编码核酮糖-1，5-二磷酸羧化酶/加氧酶（Rubisco）的超嗜热古菌，热球菌Kodakaraensis KOD 1。该基因编码的蛋白质由444个氨基酸残基组成，大小相当于以前报道的Rubiscos大亚基。KodakaraensisKOD 1的Rubisco（Pk-Rubisco）与菠菜I型Rubisco大亚基的相似性仅为51.4%，与红杜鹃II型Rubisco大亚基的相似性仅为47.3%，表明该酶不属于这两种类型。从I型和II型Rubiscos鉴定的活性位点残基是保守的。我们在大肠杆菌中表达了该基因，并获得了具有预期分子量和N-末端氨基酸序列的可溶性蛋白。重组蛋白的纯化结果表明，Pk-Rubisco为L 8型同源八聚体。Pk-Rubisco显示出最高的比活性，为19.8 × 10（3）nmol CO2固定/min/mg，在90 ℃时tau值为310 ...更多信息 degreesC，均高于任何先前表征的Rubisco。北方杂交分析表明该基因在Kodakaraensis KOD 1中有转录。此外，用P.kodakaraensis KOD 1的无细胞提取物进行的Western印迹分析清楚地表明在天然宿主细胞中存在Pk-Rubisco。为了研究天然Pk-Rubisco中小亚基的存在，进行了免疫沉淀和天然PAGE实验。当KOD 1的无细胞提取物与针对重组酶的多克隆抗体进行免疫沉淀时，检测到除了预期的PK-Rubisco大亚基以外的特异性蛋白。天然和重组的Pk-Rubiscos在非变性聚丙烯酰胺凝胶电泳上显示出相同的迁移率，表明天然的Pk-Rubiscos仅由大亚基组成。纯化的重组Pk-Rubisco的电子显微镜照片显示五角环状组装的分子。从硫酸铵溶液获得的Pk-Rubisco晶体衍射X射线超过2.8 A分辨率。衍射数据的自旋转函数表明存在相互垂直的5重轴和2重轴。沿着由凝胶过滤法估算的Pk-Rubisco的分子量，这些结果强烈地表明Pk-Rubisco是仅由大亚基组成的十聚体，具有五边形环状结构。这是第一个报告的十聚体组装的Rubisco，这被认为是不属于I型或II型Rubiscos。少

We have characterized the gene encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. The gene encoded a protein consisting of 444 amino acid residues, corresponding in size to the large subunit of previously reported Rubiscos. Rubisco of P.kodakaraensis KOD1 (Pk-Rubisco) showed only 51.4% similarity with the large subunit of type I Rubisco from spinach and 47.3% with that of type II Rubisco from Rhodospirillum rubrum, suggesting that the enzyme was not a member of either type. Active site residues identified from type I and type II Rubiscos were conserved. We expressed the gene in Escherichia coli, and we obtained a soluble protein with the expected molecular mass and N-terminal amino acid sequence. Purification of the recombinant protein revealed that Pk-Rubisco was an L8 type homo-octamer. Pk-Rubisco showed highest specific activity of 19.8 x 10(3) nmol of CO2 fixed per min/mg, and a tau value of 310 at 90 … More degreesC, both higher than any previously characterized Rubisco. Northern blot analysis demonstrated that the gene was transcribed in P.kodakaraensis KOD1. Furthermore, Western blot analysis with cell-free extract of P.kodakaraensis KOD1 clearly indicated the presence of Pk-Rubisco in the native host cells. In order to investigate the existence of small subunits in native Pk-Rubisco, immunoprecipitation and native-PAGE experiments were performed. No specific protein other than the expected large subunit of Pk-Rubisco was detected when the cell-free extracts of KOD1 were immunoprecipitated with polyclonal antibodies against the recombinant enzyme. Furthermore, native and recombinant Pk-Rubiscos exhibited identical mobilities on native-PAGE.These results indicated that native Pk-Rubisco consisted solely of large subunits. Electron micrographs of purified recombinant Pk-Rubisco displayed pentagonal ring-like assemblies of the molecules. Crystals of Pk-Rubisco obtained from ammonium sulfate solutions diffracted X-rays beyond 2.8 A resolution. The self-rotation function of the diffraction data showed the existence of 5-fold and 2-fold axes, which are located perpendicularly to each other. These results, along with the molecular mass of Pk -Rubisco estimated from gel filtration, strongly suggest that Pk -Rubisco is a decamer composed only of large subunits, with pentagonal ring-like structure. This is the first report of a decameric assembly of Rubisco, which is thought to belong to neither type I nor type II Rubiscos. Less