Anaerobic carbon dioxide fixation by microorganisms

柠檬酸裂解酶是还原三元酸循环的关键酶之一，从绿叶绿僵菌M1菌株中分离纯化出65 kDa的蛋白质，其N端序列与真核细胞中的柠檬酸裂解酶相似。我们分离到一个DNA片段，它包含两个相邻的开放阅读框ACLB(1197bp)和ACLA(1827bp)，其产物分别与人酶的N-末端和C-末端有显著的相似性。这些基因在大肠杆菌中的异源表达表明，这两个基因产物都是ATP-柠檬酸裂解酶活性所必需的。从含有aclBA的大肠杆菌无细胞提取液中纯化了重组酶，并对其进行了进一步的鉴定。经凝胶过滤，该重组酶的相对分子质量约为532-557 kDa。该酶以依赖于三磷酸腺苷、辅酶A和镁离子的方式催化柠檬酸的裂解，其中三磷酸腺苷和镁离子可分别被d三磷酸腺苷和锰离子…取代。更多。ADP和草酰乙酸酯可抑制该反应。这些性质表明，柠檬酸裂解酶依赖于细胞内的能量条件来控制循环通量。我们之前注意到在一种高度嗜热的考古菌--柯达卡拉热球菌KOD1中存在一种高活性的Rubisco。系统发育分析表明，包括PK-Rubisco在内的古生菌RuBiscos在一级结构上与已报道的I型和II型酶不同。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。KOD1无细胞提取物与重组酶多克隆抗体免疫共沉淀时，除预期的PK-Rubisco大亚基外，未检测到其他特异性蛋白。此外，天然和重组PK-RuBiscos在天然PAGE上表现出相同的迁移率。这些结果表明，天然PK-Rubisco只由大亚基组成。纯化的重组PK-Rubisco的电子显微镜显示其分子呈五角形的环状组装。从硫酸铵溶液中得到的PK-Rubisco晶体的X射线衍射分辨率超过2.8A。衍射数据的自旋函数表明，存在相互垂直的5重和2重轴。这些结果，以及凝胶过滤估计的PK-Rubisco的相对分子质量，有力地表明PK-Rubisco是一个只由大的亚基组成的十聚体，具有五角环状结构。这是首次报道了Rubisco的十聚体组装，它被认为既不属于类型I，也不属于类型II。较少

ATP-citrate lyase, one of the key enzymes of the reductive tricarboxylic acid cycle, was partially purified from Chlorobium limicola strain M1 and the N-terminal sequence of a 65-kDa protein was found to show similarity toward eukaryotic ATP-citrate lyase. We isolated a DNA fragment containing two adjacent open reading frames, aclB (1197 bp) and aclA (1827 bp), whose products showed significant similarity to the N- and C-terminal regions of the human enzyme, respectively. Heterologous expression of these genes in Escherichia coli showed that both gene products were essential for ATP-citrate lyase activity. The recombinant enzyme was purified from the cell-free extract of E.coli harboring aclBA for further characterization. The molecular mass of the recombinant enzyme was determined to be approximately 532-557 kDa by gel-filtration. The enzyme catalyzed the cleavage of citrate in an ATP-, CoA- and Mg2+-dependent manner, where ATP and Mg2+ could be replaced by dATP and Mn2+, respectively … More . ADP and oxaloacetate inhibited the reaction. These properties suggested that ATP-citrate lyase from C.limicola controlled the cycle flux depending on intracellular energy conditions. We previously noticed the presence of a highly active, Rubisco in a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. Phylogenetic analysis of Rubiscos indicated that archaeal Rubiscos, including Pk -Rubisco, were distinct from previously reported type I and type II enzymes in terms of primary structure. 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