Graduate School of Science, Osaka City University* Graduate School of Science, Osaka University, Japan** Graduate School of Science, Osaka City University, Japan***
○Kentaro Kai* Ikuko Miyahara* Noriko Nakagawa** Seiki Kuramitsu** Nobuo Kamiya***
ADP-ribose pyrophosphatase (ADPRase) from Thermus thermophilus (Tt) HB8 is a member of the nudix family proteins that distribute widely in nature and metabolize many kinds of nucleotide diphosphates. Tt-ADPRase catalyzes the divalent metal ion-dependent hydrolysis of ADP-ribose (ADPR) to AMP and ribose 5' -phosphate. The crystal structures of ADPRase have been reported including the ternary complex with divalent metal ions and ADPR (Yoshiba, S. et al. (2004) J. Biol. Chem. 279, 37163-74). The complex crystals were obtained in acetate buffer out of catalytically optimum condition pH 6-7. Two metal binding sites, MI and MII, were involved in the metal coordination scheme around ADPR in the reaction cavity. We continued to improve the ternary complex crystal with Zn (II) ions and ADPR, and succeeded to determine the structure at 1.6Å resolution using X-ray diffraction data collected at the PF. When the pH value was increased slightly to pH4.7, an additional Zn ion was newly found at the third metal binding site, MIII, interacting with the ADPR phosphate and the Glu82 carboxylate. A water molecule bridging two Zn ions at MI and MIII is a candidate of nucleophile whichi is stereochemically suitable in the inline mechanism of the nucleotide diphosphate hydrolysis. The ternary complex crystal will be a potential target for time-resolved X-ray crystallography to prove the reaction mechanism proposed, if the catalytic reaction in the crystalline state could be triggered by the pH shift toward the optimum condition pH 6-7. We are at present investigating the protocol for the pH shift in crystal, and may present more detailed reaction mechanism of ADPRase, based on the results from both of the static and time-resolved structure analyses.