Structural view of the regulatory subunit of aspartate kinase from <italic>Mycobacterium tuberculosis

doi:10.1007/s13238-011-1094-2

Prot Cell

2011, Vol. 2

Issue (9) : 745-754 https://doi.org/10.1007/s13238-011-1094-2 PMID: 21976064

RESEARCH ARTICLE

Structural view of the regulatory subunit of aspartate kinase from Mycobacterium tuberculosis

Qingzhu Yang¹, Kun Yu², Liming Yan², Yuanyuan Li¹, Cheng Chen², Xuemei Li¹(

)

1. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; 2. Structural Biology Laboratory, Tsinghua University, Beijing 100084, China

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Abstract

The aspartate kinase (AK) from Mycobacterium tuberculosis (Mtb) catalyzes the biosynthesis of aspartate family amino acids, including lysine, threonine, isoleucine and methionine. We determined the crystal structures of the regulatory subunit of aspartate kinase from Mtb alone (referred to as MtbAKβ) and in complex with threonine (referred to as MtbAKβ-Thr) at resolutions of 2.6 ? and 2.0 ?, respectively. MtbAKβ is composed of two perpendicular non-equivalent ACT domains [aspartate kinase, chorismate mutase, and TyrA (prephenate dehydrogenase)] per monomer. Each ACT domain contains two α helices and four antiparallel β strands. The structure of MtbAKβ shares high similarity with the regulatory subunit of the aspartate kinase from Corynebacterium glutamicum (referred to as CgAKβ), suggesting similar regulatory mechanisms. Biochemical assays in our study showed that MtbAK is inhibited by threonine. Based on crystal structure analysis, we discuss the regulatory mechanism of MtbAK.

Keywords Mycobacterium tuberculosis aspartate kinase crystal structure β subunit

Corresponding Author(s): Li Xuemei,Email:lixm@sun5.ibp.ac.cn

Issue Date: 01 September 2011

Cite this article:

Qingzhu Yang,Kun Yu,Liming Yan, et al. Structural view of the regulatory subunit of aspartate kinase from Mycobacterium tuberculosis[J]. Prot Cell, 2011, 2(9): 745-754.

URL:

https://academic.hep.com.cn/pac/EN/10.1007/s13238-011-1094-2
https://academic.hep.com.cn/pac/EN/Y2011/V2/I9/745

Tab.1 Data collection statistics

Fig.1
The first step in the biosynthesis of the aspartate group of amino acids, phosphorylation of aspartic acid, is catalyzed by MtbAK.

Fig.2 β.
(A) The α subunit and . subunit of MtbAK are shown by striped and dotted bars respectively. The β subunit of MtbAK was used for structural study in our work. (B) Overall structure of MtbAKβ-free monomer. The ACT1 and ACT2 domains are indicated by light pink and sand respectively. (C) Overall structure of MtbAKβ-Thr monomer. ACT1 and ACT2 domains are indicated by green and yellow respectively. Bound Thr is shown as orange spheres. (D) The structure of homodimeric MtbAKβ-Thr. The molecules are colored in green and cyan respectively. Bound Thr is shown as orange spheres. The interface, consisting of residues essential for intermolecular contact (distance less than 3.6 ?), is colored in red. (E) Elution pro?les of MtbAKβ in the presence and absence of 10 mmol/L Thr. Blue and red lines indicate in the absence and presence of 10 mmol/L Thr, respectively. Elution volumes for BSA (67.0 kDa), ovalbumin (43.0 kDa), carbonic anhydrase (29.0 kDa) and ribonuclease A (13.7 kDa) are indicated by a, b, c, and d, respectively. Data were resolved and plotted by MATLAB (http://www.mathworks.com/products/matlab).

Fig.3 βββ
(A) Superimposition of MtbAKβ-Thr monomer (green) onto CgAKβ-Thr (PDB code: 2DTJ) monomer (magenta). CgAKβ-Thr contains an extra β strand at the C terminus. Bound threonines in MtbAKβ-Thr and CgAKβ-Thr are shown as orange and magenta sticks respectively. (B) Superimposition of MtbAKβ-Thr monomer (green) onto TtAKβ-Thr (PDB code: 2DT9) monomer (lightblue). Bound threonines in MtbAKβ-Thr and TtAKβ-Thr are shown as orange and marine sticks respectively. Positions of difference are marked by dashed boxes. (C) Structure-based sequence comparison of the β subunit of aspartate kinases from , and . Secondary structure elements of the β subunit of aspartokinase from are shown at top of the alignment.

Fig.4 Thr-binding site and the enzyme activity study.
(A) Thr-binding site in MtbAKβ-Thr. Green, chain X; blue, chain Y; orange, Thr. Residues from chain Y are shown with asterisks. Number after the residue represents the residue number from β subunit. (B) Vacant Thr-binding site in MtbAKβ-free. sand, chain X; skyblue, chain Y; Residues from chain Y are shown with asterisks. Number after the residue represents the residue number from β subunit. (C) Inhibition profile of MtbAK by threonine. The histograms represent the mixture with 10 mmol/L Thr, 10 mmol/L Lys, 10 mmol/L Thr plus 10 mmol/L Lys and without inhibitor as control, respectively.

Fig.5 ββ
(A) Superposition of the inhibitor-binding units of unliganded MtbAKβ and MtbAKβ-Thr. MtbAKβ-Thr and unliganded MtbAKβ are in green and sand, respectively. The Thr molecule is depicted as a stick model. ACT1 shows the Cαn models of the Y chain (residues 1-11 and 95-168) in MtbAKβ-Thr and unliganded MtbAKβ, and ACT2 shows Cα models of the X chain (residues 12-94) in MtbAKβ-Thr and unliganded MtbAKβ. (B) Domain motion in MtbAKβ caused by Thr binding. The structures of MtbAKβ-Thr and unliganded MtbAKβ are shown in green and sand, respectively. Domain motion was resolved using DYNDOM (). A dash line demonstrates hinge axis of the movement.

Tab.2 Analysis of the dimer interface by the Protein-Protein Interaction Server (http://www.biochem.ucl.ac.uk/bsm/PP/server)

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