Abstract
ADP-forming acetyl-CoA synthetase (ACD), the novel enzyme of acetate formation and energy conservation in archaea (\({\text{Acetyl - CoA}} + {\text{ADP}} + {\text{P}}_{{\text{i}}} \rightleftarrows {\text{acetate}} + {\text{ATP}} + {\text{CoA}}\)), has been studied only in few hyperthermophilic euryarchaea. Here, we report the characterization of two ACDs with unique molecular and catalytic features, from the mesophilic euryarchaeon Haloarcula marismortui and from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. ACD from H. marismortui was purified and characterized as a salt-dependent, mesophilic ACD of homodimeric structure (166 kDa). The encoding gene was identified in the partially sequenced genome of H. marismortui and functionally expressed in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies following solubilization and refolding in the presence of salts. The ACD catalyzed the reversible ADP- and Pi-dependent conversion of acetyl-CoA to acetate. In addition to acetate, propionate, butyrate, and branched-chain acids (isobutyrate, isovalerate) were accepted as substrates, rather than the aromatic acids, phenylacetate and indol-3-acetate. In the genome of P. aerophilum, the ORFs PAE3250 and PAE 3249, which code for α and β subunits of an ACD, overlap each other by 1 bp, indicating a novel gene organization among identified ACDs. The two ORFs were separately expressed in E. coli and the recombinant subunits α (50 kDa) and β (28 kDa) were in-vitro reconstituted to an active heterooligomeric protein of high thermostability. The first crenarchaeal ACD showed the broadest substrate spectrum of all known ACDs, catalyzing the conversion of acetyl-CoA, isobutyryl-CoA, and phenylacetyl-CoA at high rates. In contrast, the conversion of phenylacetyl-CoA in euryarchaeota is catalyzed by specific ACD isoenzymes.
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Acknowledgments
We thank Prof. Dr C. Urbanke (Hannover, Germany) for determination of molecular mass of P. aerophilum ACD by analytical ultracentrifugation and Dr K. Schober (Braunschweig, Germany) for N-terminal aminoacid sequencing of H. marismortui ACD. The work was supported by grants from the Fonds der Chemischen Industrie. We also thank Dr S. DasSarma for providing access to the H. marismortui genome data at the UMBI web site http://zdna2.umbi.umd.edu/cgi-bin/blast/blast.pl and the web site URL http://zdna2.umbi.umd.edu/.
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Dedicated to Prof. Dr. Dr. h.c. mult. Hans Günter Schlegel on the occasion of his 80th birthday.
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Bräsen, C., Schönheit, P. Unusual ADP-forming acetyl-coenzyme A synthetases from the mesophilic halophilic euryarchaeon Haloarcula marismortui and from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. Arch Microbiol 182, 277–287 (2004). https://doi.org/10.1007/s00203-004-0702-4
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DOI: https://doi.org/10.1007/s00203-004-0702-4