Identification and characterization of NuhA, a novel Nudix hydrolase specific for ADP-ribose in the cyanobacterium Synechococcus sp. PCC 7002.
We cloned the gene for a novel Nudix hydrolase in the cyanobacterium Synechococcus sp. PCC 7002 and termed it nuhA. The deduced amino acid sequence of NuhA included the Nudix motif, GX(5)EX(7)RELXEEXGV, which is common to Nudix hydrolases, and in addition, a proline at the 15th amino acid from the C-terminus of the Nudix motif, which is characteristic of the subfamily of ADP-ribose pyrophosphatases. The recombinant NuhA with a hexahistidine tag was overexpressed in Escherichia coli and purified. The recombinant NuhA hydrolyzed ADP-ribose specifically among various nucleoside diphosphate derivatives. The hydrolytic activity for ADP-ribose required Mg(2+) and was optimal at pH 9.5. The V(max) and K(m) values of hydrolysis were 23.6 units mg(-1) and 0.094 mM, respectively. NuhA contained an uncharacterized domain in the C-terminal region, termed Pfam-B-3116, which is conserved in several hypothetical proteins. The mutated NuhA deficient in the Pfam-B-3116 domain failed to form the hexamers that are characteristic of NuhA, and exhibited a significantly higher K(m) value for ADP-ribose, suggesting that the Pfam-B-3116 domain might be responsible for oligomerization of NuhA and full binding affinity for ADP-ribose. These unique features suggest that NuhA is a novel type of ADP-ribose pyrophosphatase.
Okuda
Kenji
K
Graduate School of Science and Engineering, Ehime University, Bunkyo-cho, Matsuyama 790-8577, Japan.
Nishiyama
Yoshitaka
Y
Morita
Eugene H
EH
Hayashi
Hidenori
H
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D018257:Helix-Loop-Helix Motifs; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D011755:Pyrophosphatases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D010446:Peptide Fragments; D011994:Recombinant Proteins; D000246:Adenosine Diphosphate Ribose; D011755:Pyrophosphatases