Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

5A, 6A, NW12/2005G268

Crystallographic analysis of Aspartate kinase from Thermas flavus
1

Ayako Yoshida1, Takeo Tomita1, Tomohisa Kuzuyama1, Makoto Nishiyama*1
Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Introduction
Aspartate kinase (AK) catalyses the phosphorylation of
aspartic acid, which is the first step of the biosynthesis of
aspartic amino acid family, lysine, threonine, andmethionine. AK is regulated via feedback inhibition by
those end products. AK from Thermus flavus is inhibitedby threonine [1], while AK from Corynebacterium
glutamicum is concertedly inhibited by lysine andthreonine [2]. However, the mechanism of regulation ofAKs is unclear because structural information isunavailable. On the other hand , AK is an important target
for the industrial biosynthesis of amino acids, because theamino acids produced from these pathwas following thecatalytic reaction of AK are essential for mammals.
Therefore, these AKs are significant subject for bothscientific and application, however, regulatory andcatalytic mechanism have not been elucidated because nostructural information is unavailable.
AK from T. flavus is composed of two subunits, a andb, which are encoded by an in-frame overlapping gene [1]as in C. glutamicum AK and B. subtilis AK II and has
α2β2 type structure. β subunit is identical to around 160
amino acids of C-terminus of a subunit. In our previousstudies, in this α2β2 type AK, C-terminal region of αsubunit and b subunit were found to be regulatory domain,
and N-terminus region of a subunit is catalytic domainfrom chimeric AK experiment [2]. To elucidate themechanism of regulation of AK including threonine
binding site and the actions for catalytic domain, we
conducted crystallographic analysis of aspartate kinasefrom T. flavus.
Results and Discussion
The gene of T. flavus AK b subunit was obtained by
PCR amplification and it has (His)6 –tag at the Cterminal end. Amplified DNA fragment was once cloned
into pT7Blue-T-vector to confirm the nucleotide sequence
by using DSQ-2000L automatic sequencer (Shimadzu Co.,
Kyoto, Japan). Checking the nucleotide sequence, theDNA fragment was introduced into the expression vector
pET-26b(+). The construct was transformed into BL21Codon-Plus (DE3)-RIL competent cells. Ther cells weregrown in 2x YT broth in the presence of kanamycin (50mg/ml) and , chloramphenicol (30 mg/ml) at 303 K.
When the O.D. 600 reached about 0.6, protein expression
was induced y adding 0.1 mM isopropyl b-Dthiogalactopyranoside and the culture was continued for

228

Users' Report

additional 12-14 h. The cells were harvested by thecontrifugation and suspended in buffer A (20 mM TrisHCl (pH7.5)) and washed with buffer B (20 mM Tris-HCl(pH7.5), 150 mM NaCl). Suspended cells were disrupted
by sonication and the crude extract was clarified bycentrifugation (17000 rpm, 25 min). The supernatant wasmixed with Ni-resin equilibrated with buffer B containing
20 mM Imidazode. The resin was washed with the buffer
B containing 20 mM and 50 mM Imidazode in sequence,and proteins bound to the resin were eluted with thebuffer B containing 200 mM and 500 mM Imidazole.
After concentration with VIVASPIN20 , the sample wasloaded to gel filtration FPLC column, HiLoad 26/60
Superdex 75 (Amersham Pharmacia Bioscience). The
homogeneity of the purified protein was assessed bySDS-PAGE. Protein concentration was determined by themethod of Bradford using a protein assay kit (NipponBio-Rad). Final recovery of purified b subunit of T. flavus
AK was over 60 mg protein per litre of starting
culture.Screening of crystallization conditions are nowunderway.
References[1] M. Nishiyama et al., Microbiology, 141, 1211 (1995).
[2] C. Kato et al. Biochem. Biophys. Res. Commun. 316,802 (2004)* umanis@mail.ecc.u-tokyo.ac.jp