ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola is aheteromeric enzyme composed of two distinct gene products

The reductive tricarboxylic acid cycle functions as a carbon dioxide fixati on 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 fou nd to show similarity toward eukaryotic ATP-citrate lyase. A DNA fragment w as amplified with primers designed from this sequence and an internal seque nce highly conserved among eukaryotic enzymes. Using this fragment as a pro be, we isolated a DNA fragment containing two adjacent open reading frames, aclB (1197 bp) and aclA (1827 bp), whose products showed significant simil arity to the N- and C-terminal regions of the human enzyme, respectively. H eterologous expression of these genes in Escherichia coli showed that both gene products were essential for ATP-citrate lyase activity. The recombinan t 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 enzy me catalyzed the cleavage of citrate in an ATP-, CoA- and Mg2+-dependent ma nner, where ATP and Mg2+ could be replaced by dATP and Mn2+, respectively. ADP and oxaloacetate inhibited the reaction. These properties suggested tha t ATP-citrate lyase from C. limicola controlled the cycle flux depending on intracellular energy conditions. This paper provides the first direct evid ence that a bacterial ATP-citrate lyase is a heteromeric enzyme, distinct f rom mammalian enzymes.