| Budget Amount *help |
¥12,900,000 (Direct Cost: ¥12,900,000)
Fiscal Year 2000: ¥6,100,000 (Direct Cost: ¥6,100,000)
Fiscal Year 1999: ¥6,800,000 (Direct Cost: ¥6,800,000)
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| Research Abstract |
ATP-citrate lyase, one of the key enzymes of the reductive tricarboxylic acid cycle, was partially purified from Chlorobium limicola strain M1 and the N-terminal sequence of a 65-kDa protein was found to show similarity toward eukaryotic ATP-citrate lyase. 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
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. 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. We previously noticed the presence of a highly active, Rubisco in a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. Phylogenetic analysis of Rubiscos indicated that archaeal Rubiscos, including Pk -Rubisco, were distinct from previously reported type I and type II enzymes in terms of primary structure. In order to investigate the existence of small subunits in native Pk-Rubisco, immunoprecipitation and native-PAGE experiments were performed. No specific protein other than the expected large subunit of Pk -Rubisco was detected when the cell-free extracts of KOD1 were immunoprecipitated with polyclonal antibodies against the recombinant enzyme. Furthermore, native and recombinant Pk-Rubiscos exhibited identical mobilities on native-PAGE.These results indicated that native Pk-Rubisco consisted solely of large subunits. Electron micrographs of purified recombinant Pk-Rubisco displayed pentagonal ring-like assemblies of the molecules. Crystals of Pk -Rubisco obtained from ammonium sulfate solutions diffracted X-rays beyond 2.8 A resolution. The self-rotation function of the diffraction data showed the existence of 5-fold and 2-fold axes, which are located perpendicularly to each other. These results, along with the molecular mass of Pk -Rubisco estimated from gel filtration, strongly suggest that Pk-Rubisco is a decamer composed only of large subunits, with pentagonal ring-like structure. This is the first report of a decameric assembly of Rubisco, which is thought to belong to neither type I nor type II Rubiscos. Less
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