Cloning and characterization of a new member of the nudix hydrolases from human and mouse

Proteins containing the Nudix box "GX(5)EX(7)REUXEEXGU" (where U is usually Leu, Val, or lie) are Nudix hydrolases, which catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives. Here we report cloning and characterization of a human cDNA encoding a novel nudix hydrolase NUDT5 for the hydrolysis of ADP-sugars. The deduced amino acid sequence of NUDT5 con tains 219 amino acids, including a conserved Nudix hox sequence. The recomb inant NUDT5 was expressed in Escherichia coli and purified to near homogene ity. At the optimal pH of 7, the purified recombinant NUTDT5 catalyzed hydr olysis of two major substrates, ADP-ribose and ADP-mannose with K-m values of 32 and 83 mu M, respectively; the V-max for ADP-mannose was about 1.5 ti mes that with ADP-ribose. The murine NUDT5 homolog was also cloned and char acterized. mNudT5 has 81% amino acid identity to NUDT5 with catalytic activ ities similar to NUDT5 under the optimal pH of 9. Both NUDT5 and mNudT5 tra nscripts were ubiquitously expressed in tissues analyzed with preferential abundance in liver. The genomic structures of both NUDT5 and mNudT5 were de termined and located on human chromosome 10 and mouse chromosome 2, respect ively. The role of NUDT5 in maintaining levels of free ADP-ribose in cells is discussed.