Identification, purification, and characterization of an eukaryotic-like phosphopantetheine adenylyltransferase (coenzyme A biosynthetic pathway) in the hyperthermophilic archaeon Pyrococcus abyssi.
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 degrees C of the apparent Tm 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.
Armengaud
Jean
J
CEA VALRHO, DSV-DIEP, SBTN, Service de Biochimie post-génomique and Toxicologie Nucléaire, 30207 Bagnols-sur-Cèze, France. armengaud@cea.fr
Fernandez
Bernard
B
Chaumont
Valérie
V
Rollin-Genetet
Françoise
F
Finet
Stéphanie
S
Marchetti
Charles
C
Myllykallio
Hannu
H
Vidaud
Claude
C
Pellequer
Jean-Luc
JL
Gribaldo
Simonetta
S
Forterre
Patrick
P
Gans
Pierre
P
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D003065:Coenzyme A / Q000378:metabolism*; D004926:Escherichia coli; D005057:Eukaryotic Cells / Q000201:enzymology; D006639:Histidine; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019711:Pyrococcus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D006639:Histidine; D009713:Nucleotidyltransferases; C022315:pantetheine-phosphate adenylyltransferase; D003065:Coenzyme A