AMP-forming acetyl-CoA synthetase from the extremely halophilic archaeon Haloarcula marismortui: purification, identification and expression of the encoding gene, and phylogenetic affiliation.
Halophilic archaea activate acetate via an (acetate)-inducible AMP-forming acetyl-CoA synthetase (ACS), (Acetate+ATP+CoA --> Acetyl-CoA+AMP+PP(i)). The enzyme from Haloarcula marismortui was purified to homogeneity. It constitutes a 72-kDa monomer and exhibited a temperature optimum of 41 degrees C and a pH optimum of 7.5. For optimal activity, concentrations between 1 M and 1.5 M KCl were required, whereas NaCl had no effect. The enzyme was specific for acetate (100%) additionally accepting only propionate (30%) as substrate. The kinetic constants were determined in both directions of the reaction at 37 degrees C. Using the N-terminal amino acid sequence an open reading frame - coding for a 74 kDa protein - was identified in the partially sequenced genome of H. marismortui. The function of the ORF as acs gene was proven by functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies, following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione and substrates. Refolding was dependent on salt concentrations of at least 2 M KCl. The recombinant enzyme showed almost identical molecular and catalytic properties as the native enzyme. Sequence comparison of the Haloarcula ACS indicate high similarity to characterized ACSs from bacteria and eukarya and the archaeon Methanosaeta. Phylogenetic analysis of ACS sequences from all three domains revealed a distinct archaeal cluster suggesting monophyletic origin of archaeal ACS.
Bräsen
Christopher
C
Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany.
Schönheit
Peter
P
D000106:Acetate-CoA Ligase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D002384:Catalysis; D004795:Enzyme Stability; D015870:Gene Expression / Q000235:genetics*; D019614:Haloarcula marismortui / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D011189:Potassium Chloride / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity
D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D012965:Sodium Chloride; D011189:Potassium Chloride; D000106:Acetate-CoA Ligase