Identification and characterization of NuhA, a novel Nudix hydrolase specific for ADP-ribose in the cyanobacterium Synechococcus sp. PCC 7002

Abstract

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.