Structural And Functional Studies Of Human ADP-ribose Pyrophosphatase

Nudix hydrolases, a superfamily of Mg2+-requiring enzymes catalyze the hydrolysis of nucleoside diphosphates linked to other moieties, X, and contain the sequence motif or Nudix box, GX5EX7REUXEEXGU. Human NUDT5 (hNUDT5) is an ADP-ribose pyrophosphatase (ADPRase) belonging to the Nudix hydrolase superfamily and catalyzes the hydrolysis of potentially toxic substrates. It plays important roles in controlling the intracellular level of ADP-ribose (ADPR) to prevent non-enzymatic ADP-ribosylation by hydrolyzing ADPR to AMP and ribose 5'-phosphate and in sanitizing the DNA and RNA precursor pool through hydrolysis of 8-oxo-7, 8-dihydroguanine-containing nucleotides. We report here the crystal structures of hNUDT5 in apo form and in complexes with ADPR, AMP and analog AMPCPR with bound Mg2+. hNUDT5 forms a homodimer with substantial domain swapping and assumes a structure more similar to Escherichia coli ADPRases than human ADPRase NUDT9 (hNUDT9). The adenine moiety of the substrates has extensive interactions with the enzyme and particularly forms strongπ-πstacking interactions with Trp28 of one subunit and Trp46 of the other, providing the molecular basis for its high selectivity for ADP-sugars over other sugar nucleotides. Structural comparisons with two E. coli ADPRases indicate that the existence of an aromatic residue on loop L8 seems to be positively correlated with the enzymatic activity on APnA. Structural comparison with hNUDT9 provides an explanation for the activity difference on O-acetyl-ADPRs between murine NUDT5 and hNUDT9. We performed kinetic analysis of mutants of hNUDT5 to elucidate the substrate recognition and the catalytic mechanism. Structural and biochemical data also suggest that hNUDT5 may utilize a hydrolysis mechanism analogous to that of E. coli ADPRase.