Tài liệu Báo cáo khoa học: Crystal structures of the regulatory subunit of Thr-sensitive aspartate

Crystal structures of the regulatory subunit of

Thr-sensitive aspartate kinase from Thermus thermophilus

Ayako Yoshida1

, Takeo Tomita1

, Hidetoshi Kono2

, Shinya Fushinobu3

, Tomohisa Kuzuyama1 and

Makoto Nishiyama1,4

1 Biotechnology Research Center, The University of Tokyo, Japan

2 Computational Biology Group, Quantum Beam Science Directorate, Japan Atomic Energy Agency, Kyoto, Japan

3 Department of Biotechnology, The University of Tokyo, Japan

4 RIKEN SPring-8 Center, Hyogo, Japan

Aspartate kinase (AK; EC 2.7.2.4) is an enzyme that

catalyzes the first committed step, the phosphorylation

of the c-carboxyl group of aspartate, of the biosynthetic

pathway of the aspartic acid group amino acids Lys,

Thr, Ile, and Met, in microorganisms and plants. AK

is classified into two groups according to subunit orga￾nization: homo-oligomer or heterotetramer. AK from

Thermus thermophilus (TtAK), AK from C. glutami￾cum (CgAK) and AKII from Bacillus subtilis (BsAKII)

are heterotetramers containing equimolar amounts of

a-subunits and b-subunits [1–3], whereas AKIII from

Escherichia coli (EcAKIII), AKI from Arabidopsis

thaliana and AK from Methanococcus jannaschii

(MjAK) are homo-oligomers of identical subunits

[4–6]. AK of the a2b2 type is encoded by in-frame

overlapping genes, so that the amino acid sequence of

the b-subunit is identical to about 160 amino acids of

the C-terminus of the a-subunit. As seen in other

enzymes involved in the first step in amino acid bio￾synthesis, AK is regulated through feedback inhibition

Keywords

ACT domain; allosteric regulation; crystal

structure; thermostability; threonine

biosynthesis

Correspondence

M. Nishiyama, Biotechnology Research

Center, The University of Tokyo, 1-1-1

Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Fax: +81 3 5841 8030

Tel: +81 3 5841 3074

E-mail: [email protected]

(Received 3 November 2008, revised 10

March 2009, Accepted 31 March 2009)

doi:10.1111/j.1742-4658.2009.07030.x

Crystal structures of the regulatory subunit of Thr-sensitive aspartate

kinase (AK; EC 2.7.2.4) from Thermus thermophilus (TtAKb) were deter￾mined at 2.15 A˚ in the Thr-bound form (TtAKb-Thr) and at 2.98 A˚ in the

Thr-free form (TtAKb-free). Although both forms are crystallized as

dimers, the contact surface area of the dimer interface in TtAKb-free

(3200 A˚ 2

) is smaller than that of TtAKb-Thr (3890 A˚ 2

). Sedimentation

equilibrium analyzed by ultracentrifugation revealed that TtAKb is present

in equilibrium between a monomer and dimer, and that Thr binding shifts

the equilibrium to dimer formation. In the absence of Thr, an outward

shift of b-strands near the Thr-binding site (site 1) and a concomitant loss

of the electron density of the loop region between b3 and b4 near the Thr￾binding site are observed. The mechanism of regulation by Thr is discussed

on the basis of the crystal structures. TtAKb has higher thermostability

than the regulatory subunit of Corynebacterium glutamicum AK, with a dif￾ference in denaturation temperature (Tm) of 40 C. Comparison of the

crystal structures of TtAKb and the regulatory subunit of C. glutamicum

AK showed that the well-packed hydrophobic core and high Pro content

in loops contribute to the high thermostability of TtAKb.

Abbreviations

AK, aspartate kinase; BsAKII, aspartate kinase II from Bacillus subtilis; CgAK, aspartate kinase from Corynebacterium glutamicum; CgAKb,

regulatory subunit of aspartate kinase from Corynebacterium glutamicum; DSC, differential scanning calorimetry; EcAKIII, aspartate kinase III

from Escherichia coli; MAD, multiwavelength anomalous diffraction; MjAK, aspartate kinase from Methanococcus jannaschii; TtAK, aspartate

kinase from Thermus thermophilus; TtAKb, regulatory subunit of aspartate kinase from Thermus thermophilus; TtAKb-free, Thr-free

regulatory subunit of aspartate kinase from Thermus thermophilus; TtAKb-Thr, Thr-bound regulatory subunit of aspartate kinase from

Thermus thermophilus.

3124 FEBS Journal 276 (2009) 3124–3136 ª 2009 The Authors Journal compilation ª 2009 FEBS