ADP-bildende Acetyl-CoA Synthetasen aus hyperthermophilen Archaea : Molekularbiologische und biochemische Charakterisierung von neuartigen Enzymen der Acetat-Bildung und ATP-Synthese
Acetyl-CoA synthetase (ADP-forming) (ADP-ACS) represents a novel enzyme of acetate formation and energy conservation (acetyl-CoA + ADP + Pi -> acetate + ATP + CoA) in Archaea and eukaryotic protists. The only characterized ADP-ACS in Archaea, two isoenzymes from the hyperthermophile Pyrococcus furiosus, constitute 145 kDa heterotetramers (a2, b2). By using the N-terminal amino acid sequences of both subunits, which are located at different sites in the P. furiosus chromosome, the encoding genes, designated acdAI and acdBI, were identified in the genome of P. furiosus. The genes were separetely overexpressed in Escherichia coli, and the recombinant subunits were reconstituted in vitro to the active heterotetrameric enzyme. The purified recombinant enzyme showed molecular and catalytical properties very similar to those shown by ADP-ACS purified from P. furiosus. Based on significant sequence similarity of the P. furiosus genes, five open reading frames encoding putative ADP-ACS were identified in the genome of the hyperthermophilic sulfate reducing archaeon Archaeoglobus fulgidus and one open reading frame in the hyperthermophilic methanogen Methanococcus jannaschii. The ORFs constitute fusions of the homologous P. furiosus genes encoding the a and b subunits. Two ORFs, AF1211 and AF1938, of A. fulgidus, and ORF (MJ0590) of M. jannaschii were cloned and functionally overexpressed in E. coli. The purified recombinant proteins were characterized as distinctive isoenzymes of ADP-ACS with different substrate specificities. In contrast to the Pyrococcus ADP-ACS, the ADP-ACSs of Archaeoglobus and Methanococcus constitute homodimers of about 140 kDa composed of two identical 70 kDa- subunits, which represent fusions of the homologous P. furiosus a and b subunits in an ab (AF1211, MJ0590) or ba (AF1938) orientation. On the basis of sequence comparisons there were found conserved sequence motives, containing a special histidine which is also part of the succinyl-CoA synthetase (SCS) of E. coli. In a homologous reaction to the ADP-ACS, the histidine of the SCS plays an essential role as phosphoryl acceptor in the reaction. By mutation of this histidine against aspartate the ADP-ACS completely lost its catalytic activity but showed the same characteristics in chromatographic purification, an intact oligomeric structure and a similar aggregation behavior than the unmutated protein. This suggests that this histidine is likewise involved in the catalysation mechanism of the ADP-ACS.
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