Structural and functional characterization of a novel phosphatase from the Arabidopsis thaliana gene locus At1g05000.
The crystal structure of the protein product of the gene locus At1g05000, a hypothetical protein from A. thaliana, was determined by the multiple-wavelength anomalous diffraction method and was refined to an R factor of 20.4% (R(free) = 24.9%) at 3.3 A. The protein adopts the alpha/beta fold found in cysteine phosphatases, a superfamily of phosphatases that possess a catalytic cysteine and form a covalent thiol-phosphate intermediate during the catalytic cycle. In At1g05000, the analogous cysteine (Cys(150)) is located at the bottom of a positively-charged pocket formed by residues that include the conserved arginine (Arg(156)) of the signature active site motif, HCxxGxxRT. Of 74 model phosphatase substrates tested, purified recombinant At1g05000 showed highest activity toward polyphosphate (poly-P(12-13)) and deoxyribo- and ribonucleoside triphosphates, and less activity toward phosphoenolpyruvate, phosphotyrosine, phosphotyrosine-containing peptides, and phosphatidyl inositols. Divalent metal cations were not required for activity and had little effect on the reaction.
Aceti
David J
DJ
Department of Biochemistry, The Center for Eukaryotic Structural Genomics, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA.
Bitto
Eduard
E
Yakunin
Alexander F
AF
Proudfoot
Michael
M
Bingman
Craig A
CA
Frederick
Ronnie O
RO
Sreenath
Hassan K
HK
Vojtik
Frank C
FC
Wrobel
Russell L
RL
Fox
Brian G
BG
Markley
John L
JL
Phillips
George N
GN
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
D029681:Arabidopsis Proteins; C535152:At1g05000 protein, Arabidopsis; D010749:Phosphoprotein Phosphatases; D010744:Phosphoric Monoester Hydrolases