title: X-ray structure determination and deuteration of nattokinase.
abstract: 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 D2O resistant strain of Bacillus subtilis natto was obtained by successive cultivation rounds, in which the concentration of D2O 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.
authors.affiliation: Faculty of Pharmaceutical Sciences, Chiba Institute of Science, 15-8 Shiomi-cho, Cho-shi, Chiba 288-025, Japan.
authors.forename: Yasuhide
authors.initials: Y
authors.lastname: Yanagisawa
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Chatake
authors.forename: Sawa
authors.initials: S
authors.lastname: Naito
authors.forename: Tadanori
authors.initials: T
authors.lastname: Ohsugi
authors.forename: Chieko
authors.initials: C
authors.lastname: Yatagai
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sumi
authors.forename: Akio
authors.initials: A
authors.lastname: Kawaguchi
authors.forename: Kaori
authors.initials: K
authors.lastname: Chiba-Kamosida
authors.forename: Megumi
authors.initials: M
authors.lastname: Ogawa
authors.forename: Tatsumi
authors.initials: T
authors.lastname: Adachi
authors.forename: Yukio
authors.initials: Y
authors.lastname: Morimoto
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D018360:Crystallography, X-Ray; D003903:Deuterium / Q000737:chemistry*; D013381:Subtilisins / Q000737:chemistry*
chemical_list: D003903:Deuterium; D013381:Subtilisins; C053771:nattokinase
keywords: Bacillus subtilis natto; X-ray structure; deuteration; nattokinase