Cloning, expression and partial characterization of the C. elegans EEED8.8 gene product, a specific adp-ribose diphosphatase, member of nudix hydrolase family

Abstract

The C. Elegans homologue of the human YSA1 protein, EEED8.8 (Nudix6), has been expressed as a thioredoxin fusion protein in Escherichia coli. It is an ADP-sugar pyrophosphatase with similar activities towards ADP-ribose and IDP-ribose. It is a specific ADPribose (adenosine 5'-diphosphoribose) pyrophosphatase with no activities towards other nucleotides. The products of ADP-ribose hydrolysis were AMP and ribose 5-phosphate. Km and kcat values with ADP-ribose were 143.8±35.69 ìm & 18.9±2.485 ìmol/min per mg protein using ADP-ribose as substrate respectively. The optimal activity was at pH 7.2 with 10 mM Mg2+, fluoride was inhibitory, with an IC50 of 40 ìM. A major proposed function of the MutT motif proteins is to eliminate toxic nucleotide metabolites from the cell.

Key Words: ADP-ribose, C. Elegans, Nudix, EEED8.8, cloning, characterization.