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.