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HB8Okazaki, Nobuo; Adachi, Motoyasu; Tamada, Taro; Kurihara, Kazuo; Oga, Takushi*; Kamiya, Nobuo*; Kuramitsu, Seiki*; Kuroki, Ryota
Acta Crystallographica Section F, 68(1), p.49 - 52, 2012/01
Times Cited Count：2 Percentile：32.79(Biochemical Research Methods)Adachi, Motoyasu; Kuroki, Ryota; Kuramitsu, Seiki*; Oga, Takushi*
no journal, ,
ADPRase catalyzes the hydrolysis of ADP ribose to ribose 5' phosphate and AMP. The postulated role of ADPRase is to controll the cellular concentration of toxic nucleoside diphosphate devivatives. Although the crystal structure of ADPRase was solved by X-ray crystallography, it remains unclear the hydration mechanism involving hydrogen atoms by ADPRase. The objective is to reveal the detailed hydration mechanism of ADPRase using neutrron crystallogarpahy. In the symposium, we report preliminary diffraction experiment of ADPRase by neutron, preparation of large cystals in size and perdeuteration of ADPRase.
Adachi, Motoyasu; Tamada, Taro; Kurihara, Kazuo; Oga, Takushi*; Kuramitsu, Seiki*; Kuroki, Ryota
no journal, ,
ADP ribose pyrophosphatase (ADPRase) catalyzes the hydrolysis of ADP ribose to ribose 5' phosphate and AMP. In order to clarify the catalytic mechanism of ADPRase including ionization state of catalytic residues and role of water molecules on its catalytic function, recombinant ADPRase was expressed in 
and a large crystal was prepared by macro seeding. After the two months repeat of the macro seeding, a large crystal with the dimensions of 3.2
2.81.5 (13 mm
) was grown in the sodium acetate buffer (pH 5.3) containing 20% glycerol, 0.2M ammonium sulfate, and 18% PEG4000 at 20
C. Then, the crystal was soaked into the reservoir solution prepared using deuterated reagents for 20 days, and was mounted to a BIX-3 neutron diffractometer installed at the research reactor JRR-3 in JAEA. As a result of the preliminary experiments, neutron diffraction spots up to 2.1 
resolution were observed in the still images taken by 240 minutes exposure.
Adachi, Motoyasu; Tamada, Taro; Kurihara, Kazuo; Oga, Takushi*; Kuramitsu, Seiki*; Kuroki, Ryota
no journal, ,
ADP ribose pyrophosphatase (ADPRase) catalyzes the hydrolysis of ADP ribose to ribose 5' phosphate and AMP. In order to clarify the catalytic mechanism of ADPRase including ionization state of catalytic residues and role of water molecules on its catalytic function, recombinant ADPRase was expressed in and a large crystal was prepared by macro seeding. After the two months repeat of the macro seeding, a large crystal with the dimensions of 3.2 2.8 1.5 (13 mm) was grown in the sodium acetate buffer (pH5.3) containing 20% glycerol, 0.2M ammonium sulfate, and 18% PEG4000 at 20C. Then, the crystal was soaked into the reservoir solution prepared using deuterated reagents for 20 days, and was mounted to a BIX-3 neutron diffractometer installed at the research reactor JRR-3 in JAEA. As a result of the preliminary experiments, neutron diffraction spots up to 2.1 resolution were observed in the still images taken by 240 minutes exposure.
Adachi, Motoyasu; Tamada, Taro; Oga, Takushi*; Kuramitsu, Seiki*; Kuroki, Ryota
no journal, ,
ADP ribose pyrophosphatase (ADPRase) is an enzyme that catalyzes the hydrolysis of ADP ribose to ribose 5'-phosphate and AMP. Our objective is to understand the catalytic mechanism of ADPRase, including ionization state of catalytic residues and role of water molecules on the catalytic reaction, by neutron crystallography. We have attempted the perdeuteration of ADPRase using an Escherichia coli BL21(DE3) expression system and perdueterated BioExpress U-D medium (CIL Inc.). The cultivation condition was optimized to obtain maximum yield of perdueterated ADPRase. ADPRase expressed in this system was purified to homogeneity. The final yield of perdeuterated ADPRase was 38 mg from 2 liter cultivation. The perdeuterated ADPRase was then crystallized in sodium acetate buffer (pD5.5) resulting in single crystals of dimensions 0.50.30.2 mm. Optimization of the crystallization condition for X-ray and neutron crystallography is in progress.
Okazaki, Nobuo; Adachi, Motoyasu; Ohara, Takashi; Tamada, Taro; Kurihara, Kazuo; Oga, Takushi*; Kamiya, Nobuo*; Kuramitsu, Seiki*; Kuroki, Ryota
no journal, ,
no abstracts in English
