Anaerobic carbon dioxide fixation by microorganisms

微生物厌氧固定二氧化碳

• 批准号: 11450314
• 负责人: ATOMI Haruyuki
• 金额: $ 8.26万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11450314/
• 关键词: carbon dioxide fixation; Rubisco; ATP-citrate lyase; biotin; carboxylase; 超好熱菌; 始原菌; Thermococcus; archaea; 嫌気的炭酸固定; リブロースビスリン酸カルボキシラーゼ; RuBisCO

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

从石灰绿菌M1菌株中部分纯化了还原性三羧酸循环的关键酶之一ATP-柠檬酸裂解酶，发现其N-末端65-kDa的蛋白质序列与真核生物ATP-柠檬酸裂解酶相似。我们分离了一个含有两个相邻的开放阅读框的DNA片段，aclB（1197 bp）和aclA（1827 bp），其产物分别与人酶的N-和C-末端区域显示出显著的相似性。这些基因在大肠杆菌中的异源表达表明，这两个基因产物是必需的ATP-柠檬酸裂解酶活性。从含有acIBA的大肠杆菌的无细胞提取物中纯化重组酶用于进一步表征。通过凝胶过滤法测定重组酶的分子量约为532-557 kDa。该酶以ATP-、CoA-和Mg 2 +-依赖的方式催化柠檬酸的裂解，其中ATP和Mg 2+可分别被dATP和Mn 2+取代 ...更多信息 . ADP和草酰乙酸抑制该反应。这些性质表明，来自C.limicola的ATP-柠檬酸裂解酶依赖于胞内能量条件来控制循环通量。我们以前注意到存在一个高度活跃的，Rubisco在一个超嗜热古菌，Pyrococcus kodakaraensis KOD 1。Rubiscos的系统发育分析表明，古细菌Rubiscos，包括Pk-Rubisco，不同于以前报道的I型和II型酶的一级结构。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。当用针对重组酶的多克隆抗体免疫沉淀KOD 1的无细胞提取物时，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。这些结果，沿着由凝胶过滤估计的Pk-Rubisco的分子量，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。这是首次报道的十聚体组装的Rubisco，这被认为是不属于I型或II型Rubiscos。少

项目成果

S. Ezaki et al.: "Gene analysis and enzymatic properties of thermostable β-glycosidase from Pyrococcus kodakaraensis KOD1"J. Biosci. Bioeng.. 88. 130-135 (1999)

S. Ezaki 等：“Pyrococcus kodakaraensis KOD1 的热稳定性 β-糖苷酶的基因分析和酶特性”J. Biosci. 88. 130-135 (1999)

T.Tanaka et al.: "A unique chitinase with dual active sites and triple substrate binding sites from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1"Appl. Environ. Microbiol.. 65. 5338-5344 (1999)

T.Tanaka 等人：“一种独特的几丁质酶，具有来自超嗜热古菌小田火球菌 KOD1 的双重活性位点和三重底物结合位点”Appl。

Haruyuki Atomi: "Rubisco from the hyperthermophilic archaeon, Thermococcus kodakaraensis."Methods in Enzymol.. 331. 353-365 (2001)

Haruyuki Atomi：“Rubisco 来自超嗜热古菌 Thermococcus kodakaraensis。”Enzymol 中的方法.. 331. 353-365 (2001)

Haruyuki Atomi: "ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola is a heteromeric enzyme composed of two distinct gene products"Eur.J.Biochem.. 268(6). 1670-1678 (2001)

Haruyuki Atomi：“来自绿色硫细菌 Chlorobium limicola 的 ATP-柠檬酸裂解酶是一种由两种不同基因产物组成的异聚酶”Eur.J.Biochem. 268(6)。

N.Maeda, K.Kitano, T.Fukui, S.Ezaki, H.Atomi, K.Miki and T.Imanaka: "Ribulose bisphosphate carboxylase/oxygenase from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 is composed solely of large subunits and forms a pentagonal structure."J.Mol

N.Maeda、K.Kitano、T.Fukui、S.Ezaki、H.Atomi、K.Miki 和 T.Imanaka：“来自超嗜热古菌 Kodakaraensis KOD1 的核酮糖二磷酸羧化酶/加氧酶仅由大亚基组成，并形成