NMR STUDY OF PACE11 FROM THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS ABYSSI
M. Nálezková1, L.
Blanchard2, A. de Groot2, P. Gans2, D. Marion2
The analysis of the
hyperthermophilic archaeon Pyrococcus
abyssi genome showed that some proteins are highly conserved between
Archaea and Eukaryotes. 32 proteins have been listed and called the PACE
proteins (Proteins of Archaea Conserved in Eukaryotes)
[1]. One of these proteins, PACE11, we decided to study by NMR. This protein
has been biochemically characterized as a monomeric phosphopantetheine
adenylyltransferase (PPAT) involved in the Coenzyme A (CoA) biosynthesis
pathway [2]. This pathway, consisting of 5 steps, has been described in
Bacteria and Eukaryotes but not in Archaea. PPAT is involved in 4th
step of the CoA biosynthesis, catalyzing the reversible transfer of an adenylyl
group from ATP to 4‘-phosphopantetheine giving dephosphoCoA (dPCoA). So far the
3D structure of only a bacterial PPAT (from E.
coli and T. Thermophilus) has
been solved [3]. PACE11 shows very low sequence identity with these bacterial
proteins.
PACE11 project is to
get structural and dynamical information of this protein. PACE11 is PPAT of Pyrococcus abyssi, which is composed of
157 residues. In order to get enough protein for the NMR study a synthetic gene
of PACE11 (having no E. coli rare
codons) was cloned into the pET28a plasmid and the expression of the gene was
tested showing a very good production of PACE11. A 15N sample was
then prepared and the 1H-15N HSQC recorded on this sample
showed that PACE11 is well folded.
[1] Matte-Tailliez O, Zivanovic Y, Forterre P. (2000), Trends Genet 12, 533-536
[2] Armengaud J, Fernandez B, Chaumont V, Rollin-Genetet F, Finet S, Marchetti C, Myllykallio H, Vidaud C, Pellequer JL, Gribaldo S, Forterre P, Gans P. (2003), J Biol Chem 278, 31078-31087
[3] Izard T. (2002), J Mol biol 315, 487-495