Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 1670-1678 |
| Number of pages | 9 |
| Journal | European Journal of Biochemistry |
| Volume | 268 |
| Issue number | 6 |
| DOIs | |
| Publication status | Published - 2001 |
| Externally published | Yes |
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Keywords
- ATP-citrate lyase
- Chlorobium limicola
- Reductive tricarboxylic acid cycle
ASJC Scopus subject areas
- Biochemistry
Cite this
ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola is a heteromeric enzyme composed of two distinct gene products. / Kanao, Tadayoshi; Fukui, Toshiaki; Atomi, Haruyuki; Imanaka, Tadayuki.
In: European Journal of Biochemistry, Vol. 268, No. 6, 2001, p. 1670-1678.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola is a heteromeric enzyme composed of two distinct gene products
AU - Kanao, Tadayoshi
AU - Fukui, Toshiaki
AU - Atomi, Haruyuki
AU - Imanaka, Tadayuki
PY - 2001
Y1 - 2001
N2 - 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.
AB - 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.
KW - ATP-citrate lyase
KW - Chlorobium limicola
KW - Reductive tricarboxylic acid cycle
UR - http://www.scopus.com/inward/record.url?scp=0034836201&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0034836201&partnerID=8YFLogxK
U2 - 10.1046/j.1432-1327.2001.02034.x
DO - 10.1046/j.1432-1327.2001.02034.x
M3 - Article
VL - 268
SP - 1670
EP - 1678
JO - FEBS Journal
JF - FEBS Journal
SN - 1742-464X
IS - 6
ER -