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binding site of a 
-lactamase from the halophile by anomalous X-ray diffractionArai, Shigeki; Shibazaki, Chie; Shimizu, Rumi; Adachi, Motoyasu; Tamada, Taro; Tokunaga, Hiroko*; Ishibashi, Matsujiro*; Tokunaga, Masao*; Kuroki, Ryota
Kyushu Shinkurotoronko Kenkyu Senta Nempo, 2014, p.17 - 19, 2016/03
no abstracts in English
sp. AS-131 isolated from Antarctic OceanYonezawa, Yasushi*; Nagayama, Aiko*; Tokunaga, Hiroko*; Ishibashi, Matsujiro*; Arai, Shigeki; Kuroki, Ryota; Watanabe, Keiichi*; Arakawa, Tsutomu*; Tokunaga, Masao*
Protein Journal, 34(4), p.275 - 283, 2015/08
Times Cited Count：4 Percentile：10.20(Biochemistry & Molecular Biology)Nucleoside diphosphate kinase isolated from psychrophilic sp. AS-131 (ASNDK) was expressed in 
and purified to homogeneity. Comparing to mesophilic NDK isolated from 
, ASNDK exhibited highly elevated thermolability: (1) 
expression at 37
C as a denatured insoluble form, and (2) 30C lower optimum temperature of enzymatic activity. The subunit structure of ASNDK was suggested to be dimer, as in NDKs isolated from moderate halophiles.
-lactamase from the moderate halophile 
sp.560 and the discovery of a Cs-selective binding siteArai, Shigeki; Yonezawa, Yasushi*; Okazaki, Nobuo*; Matsumoto, Fumiko*; Shibazaki, Chie; Shimizu, Rumi; Yamada, Mitsugu*; Adachi, Motoyasu; Tamada, Taro; Kawamoto, Masahide*; et al.
Acta Crystallographica Section D, 71(3), p.541 - 554, 2015/03
Times Cited Count：8 Percentile：52.94(Biochemical Research Methods)The crystal structure of halophilic -lactamase from sp.560 (HaBLA) was determined using X-ray crystallography. Moreover, the locations of bound Sr
and Cs ions were identified by anomalous X-ray diffraction. The location of one Cs specific binding site was identified on HaBLA even in the presence of 9-fold molar excess of Na (90 mM Na /10 mM Cs). This Cs binding site is formed by two main-chain O atoms and an aromatic ring of a side chain of Trp. An aromatic ring of Trp interacts with Cs by the cation-
interaction. The observation of a selective and high-affinity Cs binding site provides important information that is useful for designing artificial Cs binding sites useful in bioremediation of radioactive isotopes.
sp.593Arai, Shigeki; Yonezawa, Yasushi*; Ishibashi, Matsujiro*; Matsumoto, Fumiko*; Adachi, Motoyasu; Tamada, Taro; Tokunaga, Hiroko*; Blaber, M.; Tokunaga, Masao*; Kuroki, Ryota
Acta Crystallographica Section D, 70(3), p.811 - 820, 2014/03
Times Cited Count：13 Percentile：63.81(Biochemical Research Methods)In order to clarify the structural basis of halophilic characteristics of an alkaline phosphatase derived from the moderate halophile sp.593 (HaAP), the tertiary structure of HaAP was determined to 2.1
resolution by X-ray crystallography. Structural properties of surface negative charge and core hydrophobicity are shown to be intermediate between halophile and non-halophile characteristics, and may explain the unique functional adaptation to a wide-range of salt concentration.
Adachi, Motoyasu; Arai, Shigeki; Hiromoto, Takeshi; Kuroki, Ryota
Hamon, 24(1), p.45 - 49, 2014/02
Protein structure analysis using neutron diffraction (neutron protein crystallography; NPC) is gaining greater importance in the understanding of structure and function relationships of biological macromolecules such as proteins and DNA. Current developments of neutron diffractometers installed at the JAEA research reactor and pulsed neutron source permit observation of the locations of hydrogen atoms and hydrating water molecules and help understanding of important mechanisms of chemical reactions catalyzed by biological macromolecules. Here, we introduce practical approaches of NPC including sample preparation, crystal growth, structure determination and utilization of information obtained from NPC.
Saegusa, Jun; Kurikami, Hiroshi; Yasuda, Ryo; Kurihara, Kazuo; Arai, Shigeki; Kuroki, Ryota; Matsuhashi, Shimpei; Ozawa, Takashi; Goto, Hiroaki; Takano, Takao; et al.
Health Physics, 104(3), p.243 - 250, 2013/03
Times Cited Count：3 Percentile：24.48(Environmental Sciences)After the Nuclear accident on March 2011, water discharge from many outdoor swimming pools in the Fukushima prefecture was suspended out of concern that radiocesium in the pool water would flow into farmlands. We have reviewed the existing flocculation method for decontaminating pool water and established a practical decontamination method by demonstrating the process at several pools in the Fukushima prefecture.
Niwa, Hideharu*; Saito, Makoto*; Kobayashi, Masaki*; Harada, Yoshihisa*; Oshima, Masaharu*; Moriya, Shogo*; Matsubayashi, Katsuyuki*; Nabae, Yuta*; Kuroki, Shigeki*; Ikeda, Takashi; et al.
Journal of Power Sources, 223, p.30 - 35, 2013/02
Times Cited Count：18 Percentile：49.15(Chemistry, Physical)To design non-platinum, inexpensive, but high performance carbon-based cathode catalysts for polymer electrolyte fuel cells, it is important to elucidate the active site for oxygen reduction reaction (ORR). However, it is difficult to directly identify the active site by applying conventional structural or electronic probes to such complex systems. Here, we used C 1
X-ray absorption spectroscopy (XAS) to observe electronic structure of carbon in iron phthalocyanine-based catalysts, and found a signature of edge exposure below the 
edge, whose intensity is well correlated with the ORR activity. These results demonstrate that C 1 XAS can be used to characterize the ORR activity of carbon-based cathode catalysts in terms of the edge exposure.
Kiuchi, Hisao*; Niwa, Hideharu*; Kobayashi, Masaki*; Harada, Yoshihisa*; Oshima, Masaharu*; Chokai, Masayuki*; Nabae, Yuta*; Kuroki, Shigeki*; Kakimoto, Masaaki*; Ikeda, Takashi; et al.
Electrochimica Acta, 82(1), p.291 - 295, 2012/10
Times Cited Count：14 Percentile：33.07(Electrochemistry)We study the characteristics of oxygen adsorption on metal-free carbon-based cathode catalysts derived from nitrogen-containing polyamide (PA) and nitrogen-free phenolic resin (PhRs). Electrochemical analysis and Raman spectroscopy showed higher 2-electron oxygen reduction reaction (ORR) activity and more defect sites in PA than PhRs. The increase in the amount of adsorbed oxygen in PA was also identified by oxygen adsorption isotherms. 
X-ray photoelectron spectroscopy reveals that graphite-like nitrogen contributes to oxygen adsorption and C=O components are dominant in PA. These experimental results indicate that the adsorbed C=O components near the graphite-like nitrogen can be assigned as active sites for 2-electron ORR.
1-tetrahydrocannabinolic acid (THCA) synthase, the enzyme controlling the psychoactivity of 
Shoyama, Yoshinari*; Tamada, Taro; Kurihara, Kazuo; Takeuchi, Ayako*; Taura, Futoshi*; Arai, Shigeki; Blaber, M.*; Shoyama, Yukihiro*; Morimoto, Satoshi*; Kuroki, Ryota
Journal of Molecular Biology, 423(1), p.96 - 105, 2012/10
Times Cited Count：90 Percentile：90.03(Biochemistry & Molecular Biology)1-tetrahydrocannabinolic acid (THCA) synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into THCA, the precursor of the primary psychoactive agent 1-tetrahydrocannabinol in . The structure-function relationship of THCA synthase was investigated by X-ray structure determination (2.75 resolution) and mutational analysis. Specific amino acid residues were identified in the active site of THCA synthase that are involved in the oxidative cyclization of the CBGA substrate.
Ishibashi, Matsujiro*; Uchino, Manami*; Arai, Shigeki; Kuroki, Ryota; Arakawa, Tsutomu*; Tokunaga, Masao*
Archives of Biochemistry and Biophysics, 525(1), p.47 - 52, 2012/09
Times Cited Count：7 Percentile：20.11(Biochemistry & Molecular Biology)Nucleoside diphosphate kinase (HsNDK) from Halobacterium salinarum requires salt at high concentrations for folding. A D148C mutant, in which Asp148 was replaced with Cys, was designed to enhance stability and folding in low salt solution by S-S bond. It showed increased thermal stability by about 10C in 0.2 M NaCl over the wild type HsNDK. It refolded from heat-denaturation even in 0.1 M NaCl, while the wild type required 2 M NaCl to achieve the same level of activity recovery. This enhanced refolding is due to the three S-S bonds between two basic dimeric units in the hexameric HsNDK structure. Moreover, salt concentration dependency of heat-denaturation process and refolding process of the wild type and D148C mutant HsNDKs were investigated by the circular dichroism and native-PAGE analysis.
sp. nucleoside diphosphate kinaseArai, Shigeki; Yonezawa, Yasushi; Okazaki, Nobuo; Matsumoto, Fumiko; Tamada, Taro; Tokunaga, Hiroko*; Ishibashi, Matsujiro*; Blaber, M.; Tokunaga, Masao*; Kuroki, Ryota
Protein Science, 21(4), p.498 - 510, 2012/04
Times Cited Count：15 Percentile：35.23(Biochemistry & Molecular Biology)In order to clarify the oligomer state of nucleoside diphosphate kinase (NDK) from moderately halophilic sp. 593 (HaNDK), the crystal structure of HaNDK was determined by X-ray crystallography. The crystal structures of the wild-type HaNDK and the mutant HaNDK (E134A) showed a dimer and a tetramer, respectively. The higher ordered association of proteins usually contributes to an increase in thermal stability and substrate affinity. The change in the assembly form by a minimum mutation may be an effective way for NDK to acquire molecular characteristics suited to various circumstances.
DSM3043; Both residues 134 and 136 are critical for the tetramer assemblyTokunaga, Hiroko*; Izutsu, Kenichi*; Arai, Shigeki; Yonezawa, Yasushi; Kuroki, Ryota; Arakawa, Tsutomu*; Tokunaga, Masao*
Enzyme and Microbial Technology, 46(2), p.129 - 135, 2010/02
Times Cited Count：6 Percentile：20.20(Biotechnology & Applied Microbiology)Both wild-type nucleoside diphosphate kinase from moderately halophilc (CsNDK (GNE), GNE represents Gly134-Asn135-Glu136) and mutant CsNDK (ANE), both of which have a neutral amino acid at residue 134, were found to form a dimer. These constructs contain Glu136, which may also cause steric barrier and charge repulsion. A double mutant, CsNDK (ANT), having Thr at 136 resulted in stable tetrameric assembly, supporting the above notion. A mutant CsNDK (GNT) reverted, however, to a dimer again, indicating that the introduced Ala residue at 134th in the double mutant generated a hydrophobic cluster consisting of the Ala residues and thereby stabilized dimer-dimer association of CsNDK assembly, while Gly destabilized it due to the loss of this cluster. Based on these observations, it is evident that both residues 134 and 136 contribute to the subunit assembly of CsNDK.
Adachi, Motoyasu; Ohara, Takashi; Kurihara, Kazuo; Tamada, Taro; Honjo, Eijiro; Okazaki, Nobuo; Arai, Shigeki; Shoyama, Yoshinari; Kimura, Kaname*; Matsumura, Hiroyoshi*; et al.
Proceedings of the National Academy of Sciences of the United States of America, 106(12), p.4641 - 4646, 2009/03
Times Cited Count：114 Percentile：90.49(Multidisciplinary Sciences)To further understand the catalytic mechanism and inhibitor recognition of HIV-1 protease, we need to determine the locations of key hydrogen atoms in the catalytic aspartates Asp25 and Asp125. The structure of HIV-1 protease in complex with transition-state analog KNI-272 was determined by combined neutron crystallography at 1.9 resolution and X-ray crystallography at 1.4 resolution. The resulting structural data shows that the catalytic residue Asp25 is protonated and that Asp125 is deprotonated. The proton on Asp25 makes a hydrogen bond with the carbonyl group of the allophenylnorstatine group in KNI-272. The deprotonated Asp125 bonds to the hydroxyl proton of Apns. The results provide direct experimental evidence for proposed aspects of the catalytic mechanism of HIV-1 protease; and can therefore contribute substantially to the development of specific inhibitors for therapeutic application.
Tokunaga, Hiroko*; Ishibashi, Matsujiro*; Arisaka, Fimio*; Arai, Shigeki; Kuroki, Ryota; Arakawa, Tsutomu*; Tokunaga, Masao*
FEBS Letters, 582(7), p.1049 - 1054, 2008/04
Times Cited Count：17 Percentile：39.59(Biochemistry & Molecular Biology) nucleoside diphosphate kinase (HaNDK) forms a dimeric assembly and 
NDK (PaNDK) forms a tetrameric assembly. The mutation of Glu134 to Ala in HaNDK resulted in conversion of the native dimeric structure to the tetramer assembly. Conversely, the mutation of Ala134 to Glu in PaNDK leads to conversion from tetramer to dimer assembly, indicating that a single amino acid substitution at position 134 results in an alteration of the oligomeric structure of NDK. Modeling structure of HaNDK and PaNDK, based on the crystal structure of 
NDK, suggested sufficient repulsion by Glu134 to disrupt dimer-dimer interaction to form tetramer.
Adachi, Motoyasu; Ohara, Takashi; Kurihara, Kazuo; Tamada, Taro; Honjo, Eijiro*; Okazaki, Nobuo; Arai, Shigeki; Shoyama, Yoshinari*; Matsumura, Hiroyoshi*; Adachi, Hiroaki*; et al.
no journal, ,
HIV-1 protease is a dimeric aspartic protease that cleaves the nascent polyproteins of HIV-1 and plays an essential role in viral replication. To further understand the catalytic mechanism of HIV-1 protease, we have determined the crystal structure of HIV-1 protease in complex with a transition state mimetic tripeptide inhibitor, KNI-272 to 1.9 ; resolution by neutron crystallography in combination with 1.4 ; resolution X-ray diffraction data. Our results indicates that the carbonyl group of allophenylnorstatine (Apns) in KNI-272 forms a significant hydrogen bond with protonated Asp 25, and the hydrogen atom from the hydroxyl group of Apns forms a remarkable hydrogen bond with the deprotonated Asp125. These results show direct evidence that Asp25 provides a proton to carbonyl group of substrate and Asp125 contributes to activate the attacking water molecule as a nucleophile.
Adachi, Motoyasu; Ohara, Takashi; Kurihara, Kazuo; Tamada, Taro; Honjo, Eijiro*; Okazaki, Nobuo; Arai, Shigeki; Shoyama, Yoshinari*; Matsumura, Hiroyoshi*; Sugiyama, Shigeru*; et al.
no journal, ,
In this study, we determined crystal structures of HIV-1 protease complexed with inhibitor by neutron and X-ray crystallography. Finally, we refined the structures to R-factor of 17.3% and free R-factor 20.3% by neutron crystallography and to R-factor of 10.4 % and free R-factor 12.4% by X-ray crystallography. The result shows that Asp 25 residue is protonated and Asp 125 is deprotonated. These information is important to resolve catalytic mechanism and design of new potent inhibitor.
Tamada, Taro; Honjo, Eijiro; Arai, Shigeki; Kuroki, Ryota
no journal, ,
no abstracts in English
sp.593 (HaNDK)Arai, Shigeki; Yonezawa, Yasushi; Okazaki, Nobuo; Tamada, Taro; Tokunaga, Hiroko*; Ishibashi, Matsujiro*; Tokunaga, Masao*; Kuroki, Ryota
no journal, ,
The nucleoside diphosphate kinases (NDKs) are known to have a tetrameric or hexameric oligomer structure formed by association of common dimeric components. In order to understand the oligomeric structure of NDK, the wild-type NDK from Halomonas sp. 593 (HaNDK) was determined by X-ray crystallography. The wild-type HaNDK only exhibited a dimeric form that is the same as a common dimer unit seen in other NDKs. This is the first evidence of a dimeric structure for HaNDK. To explore the effect of mutation on the assembly form of HaNDK, E134, which is a key interface residue for other tetrameric NDKs, was replaced with Ala, and the structure was determined by X-ray crystallography. Two kinds of tetrameric assemblies, Type I and Type II, appeared in the asymmetric unit of the E134A mutant HaNDK. The change from dimeric to tetrameric assembly is attributed to the removal of negative charge repulsion caused by the E134 in the wild-type HaNDK.
Adachi, Motoyasu; Arai, Shigeki; Tamada, Taro; Kuroki, Ryota; Hidaka, Koshi*; Kimura, Toru*; Kiso, Yoshiaki*
no journal, ,
To obtain information for design of inhibitor against drug resistant mutant HIV protease, we determined X-ray crystal structures of A17 type HIV-1 protease complexed with inhibitors of lopinavir and KNI-1657. The gene of A17 type HIV-1 protease was synthesized chemically, and prepared using E. coli expression system. The results showed that affinity of lopinavir to the protease was 700 times lower than that of wild-type, and KNI-1657 has 20 times higher affinity to the protease than lopinavir. The crystals of complex were obtained using PEG4000 as precipitant. The diffraction data were collected at PF and SPring-8, and the structures were refined at R-factor of 19%.
-Lactamase derived from sp.560Arai, Shigeki; Tokunaga, Hiroko*; Tamada, Taro; Yonezawa, Yasushi; Adachi, Motoyasu; Yamada, Mitsugu; Ishibashi, Matsujiro*; Tokunaga, Masao*; Kuroki, Ryota
no journal, ,
Halophilic proteins can bind various inorganic ions with its negative charges located over the molecular surface. We are investigating the molecular structure of the halophilic proteins because halophilic proteins might be used as a material capturing for the rare metals and/or the radioactive metal ions. Recently, we succeeded in X-ray crystallographic analysis of the halophilic -Lactamase (HaBLA) derived from sp.560 at 3.0 resolution. From this structural analysis, we found that the back bone structure and the positively charged active site feature of HaBLA was similar to those of non-halophilic BLA. The molecular surface of HaBLA were, however, occupied by large number of negative charges. This structural information is very useful to improve the specificity of metals such as Cs or Sr.
