Crystal structure of endo-1,4--glucanase from

Arimori, Takao*; Ito, Akihiro*; Nakazawa, Masami*; Ueda, Mitsuhiro*; Tamada, Taro

Journal of Synchrotron Radiation, 20(6), p.884 - 889, 2013/11

https://doi.org/10.1107/S0909049513021110

The saccharification process is essential for bioethanol production from woody biomass including celluloses. Cold-adapted cellulase, which has sufficient activity at low temperature ( 293 K), is capable of reducing heating costs during the saccharification process and is suitable for simultaneous saccharification and fermentation. Endo-1,4--glucanase from the earthworm Eisenia fetida (EF-EG2) belonging to glycoside hydrolase family 9 has been shown to have the highest activity at 313 K, and also retained a comparatively high activity at 283 K. The recombinant EF-EG2 was purified expressed in Pichia pastoris, and then grew needle-shaped crystals with dimensions of 0.02 0.02 1 mm. The crystals belonged to the space group P3221 with unit-cell parameters of = =136 , = 55.0 ,. The final model of EF-EG2, including 435 residues, two ions, seven crystallization reagents and 696 waters, was refined to a crystallographic -factor of 14.7% (free -factor of 16.8%) to 1.5 , resolution. The overall structure of EF-EG2 has an (/) barrel fold which contains a putative active-site cleft and a negatively charged surface. This structural information helps us understand the catalytic and cold adaptation mechanisms of EF-EG2.

Mechanistic insight into hydrolysis of oxidized nucleotide diphosphates by human NUDT5

Arimori, Takao; Yamagata, Yuriko*

Fukuoka Igaku Zasshi, 102(11), p.303 - 312, 2011/11

https://doi.org/10.15017/20446

Human NUDT5 hydrolyzes 8-oxo-dGDP into 8-oxo-dGMP and inorganic phosphate and prevents mutations caused by misincorporation of 8-oxoG into DNA. In addition, NUDT5 displays a broad substrate specificity for various modified nucleotides including 8-oxo-dADP, 8-oxo-GDP, and ADP-ribose. To understand the mechanisms of substrate recognition and catalysis by NUDT5, we have determined the crystal structure of NUDT5 complexed with 8-oxo-dGDP. A comparison of the structure with the previously reported NUDT5-ADP-ribose complex structure illustrated extremely different binding modes of these substrates. Especially, unexpectedly, the positions of two phosphates of substrate ( and phosphates) in the 8-oxo-dGDP complex are completely inverted compared to those in the ADP-ribose complex, which indicates that either the catalytic residues or the nucleophilic substitution sites of substrates are different between hydrolysis reactions of these substrates. To further investigate the catalytic mechanisms, we next determined the site of nucleophilic substitution of water for 8-oxo-dGDP and ADP-ribose by the P NMR spectra of the reaction products in O-labeled water. The results clearly showed that the sites were different between these substrates, that is, 8-oxo-dGDP was attacked by a nucleophilic water at the P, whereas ADP-ribose was at the P. Thus, we revealed the unique catalytic mechanism of NUDT5 as well as the multiple substrate recognition mechanism by structural and NMR analyses.

Crystallization and preliminary X-ray diffraction studies of the catalytic domain of a novel chitinase, a member of GH family 23, from the moderately thermophilic bacterium sp. A-471

Okazaki, Nobuo; Arimori, Takao; Nakazawa, Masami*; Miyatake, Kazutaka*; Ueda, Mitsuhiro*; Tamada, Taro

Acta Crystallographica Section F, 67(4), p.494 - 497, 2011/04

https://doi.org/10.1107/S1744309111004751

Diverse substrate recognition and hydrolysis mechanisms of human NUDT5

Arimori, Takao; Tamaoki, Haruhiko*; Nakamura, Teruya*; Kamiya, Hiroyuki*; Ikemizu, Shinji*; Takagi, Yasumitsu*; Ishibashi, Toru*; Harashima, Hideyoshi*; Sekiguchi, Mutsuo*; Yamagata, Yuriko*

no journal, , 

no abstracts in English

Crystal structure of endo-1,4--glucanase from Eisenia foetida

Arimori, Takao*; Ito, Akihiro*; Nakazawa, Masami*; Ueda, Mitsuhiro*; Tamada, Taro

no journal, , 

Crystal structures of the catalytic domain of a novel chitinase belonging to GH family 23

Arimori, Takao*; Kawamoto, Noriko*; Okazaki, Nobuo*; Nakazawa, Masami*; Miyatake, Kazutaka*; Fukamizo, Tamo*; Ueda, Mitsuhiro*; Tamada, Taro

no journal, , 

Chitin, linear -1,4-linked polymer of -acetyl-D-glucosamine (NAG), is the second abundant biopolymer in nature next to cellulose. Hydrolysis of chitin provides useful products, -acetyl-chitooligosaccharides [(NAG)] and chitooligosaccharides, which have a variety of biological functions including antibacterial activity and antitumor activity. We have previously cloned a novel chitinase gene from a moderate thermophilic strain sp. A-471 (Ra-ChiC). Ra-ChiC comprises a signal peptide, a chitin-binding domain, an interdomain linker, and a catalytic domain. The catalytic domain shares amino acid sequence homology with goose type (G-type) lysozymes and, unlike other chitinases, Ra-ChiC belongs to glycohydrolase (GH) family 23. However, Ra-ChiC does not exhibit lysozyme activity, but only chitinase activity. In this study, we aim to reveal how Ra-ChiC catalyzes the hydrolysis of chitin and why Ra-ChiC exhibits chitinase activity instead of lysozyme activity. We determined the crystal structures of the catalytic domain of Ra-ChiC (Ra-ChiC), Ra-ChiC complexed with (NAG), E141Q mutant of Ra-ChiC complexed with (NAG), E162Q mutant of Ra-ChiC, and E162Q mutant of Ra-ChiC complexed with (NAG). These structures provided us structural basis of substrate recognition mechanism and revealed that Ra-ChiC has a unique substrate-binding site including a tunnel-shaped cavity, which determines the substrate specificity. In addition, we also carried out a mutation analysis of acidic amino acid residues located at the active site. As a result, we found that not only a highly conserved Glu141 but also Asp226 located at the roof of the tunnel have quite important roles in catalysis.