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Journal of Bacteriology, November 2004, p. 7754-7762, Vol. 186, No. 22
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.22.7754-7762.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification and Functional Verification of Archaeal-Type Phosphoenolpyruvate Carboxylase, a Missing Link in Archaeal Central Carbohydrate Metabolism

Thijs J. G. Ettema,1* Kira S. Makarova,2 Gera L. Jellema,1 Hinco J. Gierman,1 Eugene V. Koonin,2 Martijn A. Huynen,3 Willem M. de Vos,1 and John van der Oost1

Laboratory of Microbiology, Agrotechnology and Food Sciences, Wageningen University, Wageningen ,1 Nijmegen Center for Molecular Life Sciences, Center for Molecular and Biomolecular Informatics, Nijmegen, The Netherlands,3 National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland2

Received 10 May 2004/ Accepted 16 August 2004

Despite the fact that phosphoenolpyruvate carboxylase (PEPC) activity has been measured and in some cases even purified from some Archaea, the gene responsible for this activity has not been elucidated. Using sensitive sequence comparison methods, we detected a highly conserved, uncharacterized archaeal gene family that is distantly related to the catalytic core of the canonical PEPC. To verify the predicted function of this archaeal gene family, we cloned a representative from the hyperthermophilic acidophile Sulfolobus solfataricus and functionally produced the corresponding enzyme as a fusion with the Escherichia coli maltose-binding protein. The purified fusion protein indeed displayed highly thermostable PEPC activity. The structural and biochemical properties of the characterized archaeal-type PEPC (atPEPC) from S. solfataricus are in good agreement with previously reported biochemical analyses of other archaeal PEPC enzymes. The newly identified atPEPC, with its distinct properties, constitutes yet another example of the versatility of the enzymes of the central carbon metabolic pathways in the archaeal domain.

* Corresponding author. Mailing address: Laboratory of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands. Phone: 31(0)317-483110. Fax: 31(0)317-483829. E-mail: thijs.ettema{at}wur.nl.

Journal of Bacteriology, November 2004, p. 7754-7762, Vol. 186, No. 22
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.22.7754-7762.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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