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J. Biol. Chem., Vol. 278, Issue 33, 31078-31087, August 15, 2003

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Identification, Purification, and Characterization of an Eukaryotic-like Phosphopantetheine Adenylyltransferase (Coenzyme A Biosynthetic Pathway) in the Hyperthermophilic Archaeon Pyrococcus abyssi^*

Jean Armengaud  , Bernard Fernandez , Valérie Chaumont , Françoise Rollin-Genetet , Stéphanie Finet ¶, Charles Marchetti , Hannu Myllykallio ||, Claude Vidaud , Jean-Luc Pellequer , Simonetta Gribaldo || **, Patrick Forterre || and Pierre Gans 

From the CEA VALRHO, DSV-DIEP, SBTN, Service de Biochimie post-génomique and Toxicologie Nucléaire, 30207 Bagnols-sur-Cèze, France, the ^¶European Synchrotron Radiation Facility, BP 220, 38043 Grenoble, France, the ^||Institut de Genetique et Microbiologie, UMR CNRS 8621, Universite Paris-Sud, 91405 Orsay, France, and the Institut de Biologie Structurale CEA-CNRS-UJF, Jean-Pierre Ebel, UMR 5075, Laboratoire de Resonance Magnetique Nucleaire, 38027 Grenoble, France

Although coenzymeA (CoA) is essential in numerous metabolic pathways in all living cells, molecular characterization of the CoA biosynthetic pathway in Archaea remains undocumented. Archaeal genomes contain detectable homologues for only three of the five steps of the CoA biosynthetic pathway characterized in Eukarya and Bacteria. In case of phosphopantetheine adenylyltransferase (PPAT) (EC 2.7.7.3), the putative archaeal enzyme exhibits significant sequence similarity only with its eukaryotic homologs, an unusual situation for a protein involved in a central metabolic pathway. We have overexpressed in Escherichia coli, purified, and characterized this putative PPAT from the hyperthermophilic archaeon Pyrococcus abyssi (PAB0944). Matrix-assisted laser desorption ionization-time of flight mass spectrometry and high performance liquid chromatography measurements are consistent with the presence of a dephospho-CoA (dPCoA) molecule tightly bound to the polypeptide. The protein indeed catalyzes the synthesis of dPCoA from 4'-phosphopantetheine and ATP, as well as the reverse reaction. The presence of dPCoA stabilizes PAB0944, as it induces a shift from 76 to 82 °C of the apparent T_m measured by differential scanning microcalorimetry. Potassium glutamate was found to stabilize the protein at 400 mM. The enzyme behaves as a monomeric protein. Although only distantly related, secondary structure prediction indicates that archaeal and eukaryal PPAT belong to the same nucleotidyltransferase superfamily of bacterial PPAT. The existence of operational proteins highly conserved between Archaea and Eukarya involved in a central metabolic pathway challenge evolutionary scenarios in which eukaryal operational proteins are strictly of bacterial origin.

Received for publication, February 24, 2003 , and in revised form, May 7, 2003.

^* This work was supported in part by a grant from the Centre National de la Recherche Scientifique (Program Protéomique et Génie des Protéines, Appel à propositions 2001). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** Supported by a grant from the Association de la Recherche sur le Cancer.

To whom correspondence should be addressed: CEA VALRHO, DSV-DIEP, SBTN, Service de Biochimie post-génomique and Toxicologie Nucléaire, Marcoule, BP 17171, F-30207 Bagnols-sur-Cèze cedex, France. Tel.: 33-4-66-79-68-02; Fax: 33-4-66-79-19-05; E-mail: armengaud{at}cea.fr.

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