Open AccessX‐ray structure determination and deuteration of nattokinase
Author(s) -
Yanagisawa Yasuhide,
Chatake Toshiyuki,
Naito Sawa,
Ohsugi Tadanori,
Yatagai Chieko,
Sumi Hiroyuki,
Kawaguchi Akio,
ChibaKamosida Kaori,
Ogawa Megumi,
Adachi Tatsumi,
Morimoto Yukio
Publication year - 2013
Publication title -
journal of synchrotron radiation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.172
H-Index - 99
ISSN - 1600-5775
DOI - 10.1107/s0909049513020700
Subject(s) - nattokinase , subtilisin , bacillus subtilis , substrate (aquarium) , enzyme , chemistry , protein secondary structure , biochemistry , biology , fermentation , bacteria , ecology , genetics
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 2 O resistant strain of Bacillus subtilis natto was obtained by successive cultivation rounds, in which the concentration of D 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.