Characterization of the Arabidopsis thaliana Arath;CDC25 dual-specificity tyrosine phosphatase.
CDC25 enzymes are dual-specificity phosphatases involved in the regulation of the cell cycle. No CDC25 enzymes have been described in higher plant organisms. We report here the characterization of an Arabidopsis thaliana CDC25 enzyme, constituted by a sole catalytic domain and devoid of the N-terminal regulatory region found in the human CDC25. We describe the recombinant expression in Escherichia coli of the Arath;CDC25 and its purification for activity assay and structure determination by NMR. The recombinant enzyme has a tyrosine phosphatase activity towards an artificial substrate, a NMR characterization equally concludes to its correct folding. The secondary structure of the protein was predicted on the basis of the assigned chemical shift of (1)H, (15)N, and (13)C backbone atoms of the protein. The presence of a metal ion in the C-terminus of this new protein points to a zinc finger, and sequence homology indicates that this new structural element might be conserved in related plant homologs.
Landrieu
Isabelle
I
UMR 8525 CNRS-Lille2, Institut de Biologie de Lille/Pasteur Institute of Lille, 59019 Lille Cedex, France. isabelle.landrieu@ibl.fr
Hassan
Sahar
S
Sauty
Mathieu
M
Dewitte
Frédérique
F
Wieruszeski
Jean-Michel
JM
Inzé
Dirk
D
De Veylder
Lieven
L
Lippens
Guy
G
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D001120:Arginine; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D017124:Conserved Sequence; D003545:Cysteine; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine; D020687:cdc25 Phosphatases / Q000378:metabolism*
D029681:Arabidopsis Proteins; D003062:Codon; D017931:DNA Primers; D011994:Recombinant Proteins; D012694:Serine; D001120:Arginine; D017027:Protein Tyrosine Phosphatases; D020687:cdc25 Phosphatases; D003545:Cysteine