ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola is a heteromeric enzyme composed of two distinct gene products.
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 Mg(2+)-dependent manner, where ATP and Mg(2+) could be replaced by dATP and Mn(2+), 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.
Kanao
T
T
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Japan.
Fukui
T
T
Atomi
H
H
Imanaka
T
T
D001275:ATP Citrate (pro-S)-Lyase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D019414:Chlorobi / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D001275:ATP Citrate (pro-S)-Lyase