T. Kanao et al., ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola is aheteromeric enzyme composed of two distinct gene products, EUR J BIOCH, 268(6), 2001, pp. 1670-1678
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.