Abstract
X-ray structure determination and deuteration of nattokinase were performed to facilitate neutron crystallographic analysis. Nattokinase (NK) is a strong fibrinolytic enzyme, which is produced in abundance by Bacillus subtilis natto. Although NK is a member of the subtilisin family, it displays different substrate specificity when compared with other subtilisins. The results of molecular simulations predict that hydrogen arrangements around Ser221 at the active site probably account for the substrate specificity of NK. Therefore, neutron crystallographic analysis should provide valuable information that reveals the enzymatic mechanism of NK. In this report, the X-ray structure of the non-hydrogen form of undeuterated NK was determined, and the preparation of deuterated NK was successfully achieved. The non-hydrogen NK structure was determined at 1.74 Å resolution. The three-dimensional structures of NK and subtilisin E from Bacillus subtilis DB104 are near identical. Deuteration of NK was carried out by cultivating Bacillus subtilis natto in deuterated medium. The D{sub 2}O resistant strain of Bacillus subtilis natto was obtained by successive cultivation rounds, in which the concentration of D{sub 2}O in the medium was gradually increased. NK was purified from the culture medium and its activity was confirmed by the fibrin plate method. The results lay the framework
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Yanagisawa, Yasuhide;
[1]
Chatake, Toshiyuki;
[2]
Naito, Sawa;
Ohsugi, Tadanori;
Yatagai, Chieko;
Sumi, Hiroyuki;
[3]
Kawaguchi, Akio;
[2]
Chiba-Kamosida, Kaori;
[4]
Ogawa, Megumi;
Adachi, Tatsumi;
[1]
Morimoto, Yukio
[2]
- Chiba Institute of Science, 15-8 Shiomi-cho, Cho-shi, Chiba 288-025 (Japan)
- Kyoto University, Asashironishi 2, Kumatori, Osaka 590-0494 (Japan)
- Kurashiki University of Science and the Arts, 2640 Nishinoura, Tsurajima-cho, Kurashiki, Okayama 712-8505 (Japan)
- Nippon Advanced Technology Co. Ltd, J-PARC, 2-4 Shirane Shirakata, Tokai, Ibaraki 319-1195 (Japan)
Citation Formats
Yanagisawa, Yasuhide, Chatake, Toshiyuki, Naito, Sawa, Ohsugi, Tadanori, Yatagai, Chieko, Sumi, Hiroyuki, Kawaguchi, Akio, Chiba-Kamosida, Kaori, Ogawa, Megumi, Adachi, Tatsumi, and Morimoto, Yukio.
X-ray structure determination and deuteration of nattokinase.
Denmark: N. p.,
2013.
Web.
doi:10.1107/S0909049513020700.
Yanagisawa, Yasuhide, Chatake, Toshiyuki, Naito, Sawa, Ohsugi, Tadanori, Yatagai, Chieko, Sumi, Hiroyuki, Kawaguchi, Akio, Chiba-Kamosida, Kaori, Ogawa, Megumi, Adachi, Tatsumi, & Morimoto, Yukio.
X-ray structure determination and deuteration of nattokinase.
Denmark.
https://doi.org/10.1107/S0909049513020700
Yanagisawa, Yasuhide, Chatake, Toshiyuki, Naito, Sawa, Ohsugi, Tadanori, Yatagai, Chieko, Sumi, Hiroyuki, Kawaguchi, Akio, Chiba-Kamosida, Kaori, Ogawa, Megumi, Adachi, Tatsumi, and Morimoto, Yukio.
2013.
"X-ray structure determination and deuteration of nattokinase."
Denmark.
https://doi.org/10.1107/S0909049513020700.
@misc{etde_22432706,
title = {X-ray structure determination and deuteration of nattokinase}
author = {Yanagisawa, Yasuhide, Chatake, Toshiyuki, Naito, Sawa, Ohsugi, Tadanori, Yatagai, Chieko, Sumi, Hiroyuki, Kawaguchi, Akio, Chiba-Kamosida, Kaori, Ogawa, Megumi, Adachi, Tatsumi, and Morimoto, Yukio}
abstractNote = {X-ray structure determination and deuteration of nattokinase were performed to facilitate neutron crystallographic analysis. Nattokinase (NK) is a strong fibrinolytic enzyme, which is produced in abundance by Bacillus subtilis natto. Although NK is a member of the subtilisin family, it displays different substrate specificity when compared with other subtilisins. The results of molecular simulations predict that hydrogen arrangements around Ser221 at the active site probably account for the substrate specificity of NK. Therefore, neutron crystallographic analysis should provide valuable information that reveals the enzymatic mechanism of NK. In this report, the X-ray structure of the non-hydrogen form of undeuterated NK was determined, and the preparation of deuterated NK was successfully achieved. The non-hydrogen NK structure was determined at 1.74 Å resolution. The three-dimensional structures of NK and subtilisin E from Bacillus subtilis DB104 are near identical. Deuteration of NK was carried out by cultivating Bacillus subtilis natto in deuterated medium. The D{sub 2}O resistant strain of Bacillus subtilis natto was obtained by successive cultivation rounds, in which the concentration of D{sub 2}O in the medium was gradually increased. NK was purified from the culture medium and its activity was confirmed by the fibrin plate method. The results lay the framework for neutron protein crystallography analysis.}
doi = {10.1107/S0909049513020700}
journal = []
issue = {Pt 6}
volume = {20}
journal type = {AC}
place = {Denmark}
year = {2013}
month = {Nov}
}
title = {X-ray structure determination and deuteration of nattokinase}
author = {Yanagisawa, Yasuhide, Chatake, Toshiyuki, Naito, Sawa, Ohsugi, Tadanori, Yatagai, Chieko, Sumi, Hiroyuki, Kawaguchi, Akio, Chiba-Kamosida, Kaori, Ogawa, Megumi, Adachi, Tatsumi, and Morimoto, Yukio}
abstractNote = {X-ray structure determination and deuteration of nattokinase were performed to facilitate neutron crystallographic analysis. Nattokinase (NK) is a strong fibrinolytic enzyme, which is produced in abundance by Bacillus subtilis natto. Although NK is a member of the subtilisin family, it displays different substrate specificity when compared with other subtilisins. The results of molecular simulations predict that hydrogen arrangements around Ser221 at the active site probably account for the substrate specificity of NK. Therefore, neutron crystallographic analysis should provide valuable information that reveals the enzymatic mechanism of NK. In this report, the X-ray structure of the non-hydrogen form of undeuterated NK was determined, and the preparation of deuterated NK was successfully achieved. The non-hydrogen NK structure was determined at 1.74 Å resolution. The three-dimensional structures of NK and subtilisin E from Bacillus subtilis DB104 are near identical. Deuteration of NK was carried out by cultivating Bacillus subtilis natto in deuterated medium. The D{sub 2}O resistant strain of Bacillus subtilis natto was obtained by successive cultivation rounds, in which the concentration of D{sub 2}O in the medium was gradually increased. NK was purified from the culture medium and its activity was confirmed by the fibrin plate method. The results lay the framework for neutron protein crystallography analysis.}
doi = {10.1107/S0909049513020700}
journal = []
issue = {Pt 6}
volume = {20}
journal type = {AC}
place = {Denmark}
year = {2013}
month = {Nov}
}