ABSTRACT
ADP-ribose pyrophosphatase (ADPRase), a member of the nudix protein family, catalyzes the hydrolysis of ADP-ribose to AMP and ribose 5'-phosphate. We have determined the crystal structure of ADPRase from Thermus thermophilus HB8 (TtADPRase). We performed kinetic analysis of mutants of TtADPRase to elucidate the substrate recognition and the catalytic mechanism. Our results suggest that interactions responsible for the substrate recognition are located at the terminal moieties of the substrate. The adenine moiety is recognized by Ile-19 and the main chain carbonyl group of Glu-29 and/or Gly-104. The terminal ribose moiety is recognized by the sum of some weak interactions with multiple residues that are close in space. Glu-82 and Glu-86, conserved in the nudix motif, were previously shown to be essential for catalysis. Mutation of these residues shows that the dependence of kcat on pH is almost the same as that of the wild-type enzyme. Results suggest that Glu-82 and Glu-86 are essential for catalysis but unlikely to act as a catalytic base. In the crystal structure, each acidic residue coordinates with a metal ion. Furthermore, a water molecule coordinates between these two metals. Our results suggest a two-metal ion mechanism for the catalysis of ADPRase in which a water molecule is activated to act as a nucleophile by the cations coordinated by Glu-82 and Glu-86. Arg-54, Glu-70, Arg-81, and Glu-85 are predicted to support this nucleophilic attack on the alpha-phosphate of the substrate. Interestingly, ADPRase displays differences in the substrate recognition and the catalytic mechanism from the models proposed for other nudix proteins. Our results highlight the diversity within the nudix protein family in terms of substrate recognition and catalysis.
