Cloning and characterization of the first member of the Nudix family from Arabidopsis thaliana.
The sequence motif commonly called a Nudix box, represented by (GX(5)EX(7)REVXEEXGU) is the marker of a widely distributed family of enzymes that catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives. Here we describe the cloning and characterization of an Arabidopsis thaliana cDNA encoding a Nudix hydrolase that degrades NADH. The deduced amino acid sequence of AtNUDT1 contains 147 amino acids. The recombinant AtNUDT1 was expressed in Escherichia coli and purified. In the presence of Mn(2+) and the optimal pH of 7. 0, the recombinant AtNUDT1 catalyzed the hydrolysis of NADH with a K(m) value of 0. 36 mm. A V(max) of 12. 7 units mg (-1) for NADH was determined. The recombinant AtNUDT1 migrated as a dimer on a gel filtration column. Biochemical analysis of recombinant AtNUDT1 indicated that the first characterized member of the Nudix family from A. thaliana is a NADH pyrophosphatase.
Dobrzanska
Marta
M
Departament of Plant Biochemistry, Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warsaw, Poland. martad@ibb.waw.pl
Szurmak
Blanka
B
Wyslouch-Cieszynska
Aleksandra
A
Kraszewska
Elzbieta
E
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D007700:Kinetics; D008969:Molecular Sequence Data; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D029681:Arabidopsis Proteins; D011994:Recombinant Proteins; C471296:NUDT1 protein, Arabidopsis; D011755:Pyrophosphatases