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 Ile) 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 contains 219 amino acids, including a conserved Nudix box sequence. The recombinant NUDT5 was expressed in Escherichia coli and purified to near homogeneity. At the optimal pH of 7, the purified recombinant NUDT5 catalyzed hydrolysis of two major substrates ADP-ribose and ADP-mannose with K(m) values of 32 and 83 microM, respectively; the V(max) for ADP-mannose was about 1.5 times that with ADP-ribose. The murine NUDT5 homolog was also cloned and characterized. mNudT5 has 81% amino acid identity to NUDT5 with catalytic activities similar to NUDT5 under the optimal pH of 9. Both NUDT5 and mNudT5 transcripts were ubiquitously expressed in tissues analyzed with preferential abundance in liver. The genomic structures of both NUDT5 and mNudT5 were determined and located on human chromosome 10 and mouse chromosome 2, respectively. The role of NUDT5 in maintaining levels of free ADP-ribose in cells is discussed.
Yang
H
H
Department of Microbiology and Molecular Genetics and the Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.
Slupska
M M
MM
Wei
Y F
YF
Tai
J H
JH
Luther
W M
WM
Xia
Y R
YR
Shih
D M
DM
Chiang
J H
JH
Baikalov
C
C
Fitz-Gibbon
S
S
Phan
I T
IT
Conrad
A
A
Miller
J H
JH
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000247:Adenosine Diphosphate Sugars / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002874:Chromosome Mapping; D002879:Chromosomes, Human, Pair 10; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D005810:Multigene Family; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics; D010802:Phylogeny; D011755:Pyrophosphatases / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000247:Adenosine Diphosphate Sugars; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C406809:adenosine diphosphate-mannose; D000246:Adenosine Diphosphate Ribose; D010744:Phosphoric Monoester Hydrolases; C403715:NUDT5 protein, human; D011755:Pyrophosphatases; C052211:mutT protein, E coli