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AMP-forming acetyl-CoA synthetase from the extremely halophilic archaeon Haloarcula marismortui: purification, identification and expression of the encoding gene, and phylogenetic affiliation

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Abstract

Halophilic archaea activate acetate via an (acetate)-inducible AMP-forming acetyl-CoA synthetase (ACS), (Acetate + ATP + CoA → Acetyl-CoA + AMP + PPi). The enzyme from Haloarcula marismortui was purified to homogeneity. It constitutes a 72-kDa monomer and exhibited a temperature optimum of 41°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°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.

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Acknowledgements

We thank Dr. R. Schmid (Osnabrück, Germany) for N-terminal amino acid sequencing of H. marismortui ACS. We also thank Dr. S. DasSarma for getting 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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  1. Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 1-9, 24118, Kiel, Germany

    Christopher Bräsen & Peter Schönheit

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  1. Christopher Bräsen
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  2. Peter Schönheit
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Correspondence to Peter Schönheit.

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Communicated by G. Antranikian

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Bräsen, C., Schönheit, P. AMP-forming acetyl-CoA synthetase from the extremely halophilic archaeon Haloarcula marismortui: purification, identification and expression of the encoding gene, and phylogenetic affiliation. Extremophiles 9, 355–365 (2005). https://doi.org/10.1007/s00792-005-0449-0

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