ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola is a heteromeric enzyme composed of two distinct gene products
ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola is a heteromeric enzyme composed of two distinct gene products
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DOI：
10.1046/j.1432-1033.2001.02034.x
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发表时间：
2001-03-01
期刊：
影响因子：
--
通讯作者：
Imanaka, T
中科院分区：
文献类型：
--
作者：
Kanao, T;Fukui, T;Imanaka, T
还原性三羧酸循环在绿色硫细菌石灰绿菌中作为二氧化碳固定途径发挥作用。柠檬酸裂解酶是这一循环的关键酶之一。limicola菌株M1和65-kDa蛋白的N-末端序列被发现显示出与真核ATP-柠檬酸裂解酶的相似性。用从该序列和在真核酶中高度保守的内部序列设计的引物扩增DNA片段。以该片段为探针，我们分离出一个含有两个相邻开放阅读框aclB（1197 bp）和aclA（1827 bp）的DNA片段，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。从大肠杆菌的无细胞提取物中纯化了重组酶。大肠杆菌进行进一步表征。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盐的裂解，其中ATP和Mg 2+可分别被dATP和Mn 2+取代。ADP和草酰乙酸抑制该反应。这些性质表明，C. limicola根据细胞内能量条件控制循环通量。本文提供了第一个直接的证据表明，细菌ATP-柠檬酸裂解酶是一种异聚酶，不同于哺乳动物的酶。
The reductive tricarboxylic acid cycle functions as a carbon dioxide fixation pathway in the green sulfur bacterium, Chlorobium limicola. ATP-citrate lyase, one of the key enzymes of this cycle, was partially purified from C. limicola strain M1 and the N-terminal sequence of a 65-kDa protein was found to show similarity toward eukaryotic ATP-citrate lyase. A DNA fragment was amplified with primers designed from this sequence and an internal sequence highly conserved among eukaryotic enzymes. Using this fragment as a probe, 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. 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. This paper provides the first direct evidence that a bacterial ATP-citrate lyase is a heteromeric enzyme, distinct from mammalian enzymes.