Cloning and characterization of an Arabidopsis thaliana Nudix hydrolase homologous to the mammalian GFG protein.
In a search for a plant antimutator MutT protein, an Arabidopsis thaliana Nudix hydrolase with homology to the mammalian GFG protein was expressed as a hexahistidine fusion polypeptide in Escherichia coli and purified to homogeneity. Unlike the GFG protein, the A. thaliana homolog could not complement the mutT mutation in a MutT-deficient E. coli strain nor was it able to hydrolyze 8-oxo-dGTP, the main substrate of the MutT protein. Instead the recombinant protein hydrolyzed a variety of nucleoside diphosphate derivatives showing a preference for ADP-ribose, with Km and k(cat) values of 1.2 mM and 2.7 s(-1) respectively. The products of ADP-ribose hydrolysis were AMP and ribose-5-phosphate. The optimal activity was at alkaline pH (8.5) with Mg2+ (5 mM) ions as the cofactor. The protein exists as a dimmer in solution.
Olejnik
Kamil
K
The Department of Plant Biochemistry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warsaw, Poland.
Kraszewska
Elzbieta
E
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics*; D015153:Blotting, Western; D003001:Cloning, Molecular; D017931:DNA Primers; D003848:Deoxyguanine Nucleotides / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000235:genetics; D005816:Genetic Complementation Test; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011755:Pyrophosphatases / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization; D000096964:Nudix Hydrolases
D000246:Adenosine Diphosphate Ribose; D017931:DNA Primers; D003848:Deoxyguanine Nucleotides; D029968:Escherichia coli Proteins; D011755:Pyrophosphatases; D000096964:Nudix Hydrolases; C078206:8-oxodeoxyguanosine triphosphate; C052211:mutT protein, E coli