Photon Factory Activity Report 2014 #32 (2015) B

BL-17A, NW12A, NE3A/2012G576

Structural basis for substrate setereospecificity of D-threo-3-hydroxyaspartate
dehydratase from Delftia sp. HT23
Yu Matsumoto1, Yoshiaki Yasutake2,*, Tomohiro Tamura2, Masaru Wada1
Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan2
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology(AIST), Sapporo 062-8517, Japan1

1 Introduction
3-Hydroxyaspartate and its derivatives are known to be
useful as competitive blockers of excitatory glutamate
transporters of the mammalian central nervous system. 3Hydroxyaspartate has two chiral carbon atoms, and thus
there are four stereoisomers, i.e., D-threo-3D-THA),
L-threo-3hydroxyaspartate(2R,3R;hydroxyaspartate(2S,3S;L-THA),
D-erythro-3hydroxyaspartate (2R,3S; D-EHA), and L-erythro-3hydroxyaspartate (2S,3R; L-EHA). It is very difficult tosynthesize individually by synthetic chemistry approach.
D-threo-3-Hydroxyaspartate dehydratase (D-THA DH;
EC 4.3.1.27) [1] isolated from Delftia sp. HT23 is a novel
PLP-containing enzyme that shows dehydratase activity
against D-THA, L-THA, and L-EHA [1]. Therefore, DTHA DH is useful to produce optically pure 3DL-racemic
3hydroxyaspartate
isomers
fromhydroxyaspartate. To elucidate the substrate recognitionand catalytic mechanism regarding enantio-selectivity, we
have determined the crystal structures of D-THA DH and
its inactive mutant in complex with various substrates andinhibitors [2, 3].
2 Experiment
Recombinant His-tagged D-THA DH and its mutants
were overexpressed by Rhodococcus erythropolis L88
cells, and were purified by using Ni-affinity column.
Bipyramid-shaped single crystals were obtained byhanging-drop vapor diffusion method at 20°C, using thereservoir solution containing 0.1 M Tris-HCl, pH 8.5, 0.2
M MgCl2, and 10–18% PEG 3350. For the preparation ofsubstrate/inhibitor complexes, molar excess ofsubstrate/inhibitors were added to the crystallization
sample before crystallization setup. X-ray diffraction data
sets were collected on the beamline NW12A, NE3A andBL-17A at Photon Factory (PF), by using the CCD
detector. All the crystals of the D-THA DH and its
mutants were isomorphous: the space group I4122 with
unit-cell dimensions of approximately a = b = 157, and c= 157 Å. The diffraction images were processed with theHKL2000
programpackage.
Singlewavelength
anomalous diffraction (SAD) phasing was performed tobuild the first atomic model of D-THA DH with theprogram SHELXC/D/E, using the anomalous signal ofbromide atom as previously described [2]. Model
refinement was performed with the program REFMAC5.
Structures of D-THA DH analyzed in this study was listed
in Table 1.

Table 1: List of D-THA DH crystal structures reported inthis study.
Resolution
PDB
EnzymeLigand(Å)
ID
Wild-type
2.30
3WQF
Wild-type
Mg
1.50
3WQC
Wild-typeMg, D-EHA
1.50
3WQD
C353A
1.55
3WQG
H351A
Mg
1.70
4PB3Mg, 2-amino
maleic acid
H351A
1.80
4PB4(reactionintermediate)
H351A
Mg, L-EHA
1.90
4PB5

3 Results and Discussion
Total of seven crystal structures of wild-type andpoorly active mutant of D-THA DH were determined at
resolution ranging 1.5-2.30 Å, with and without various
substrate/inhibitors. The asymmetric unit contains ahomodimer formed by a head-to-tail association of two
polypeptide chains of D-THA DH. The structural analysis
revealed that the D-THA DH belongs to fold type III
family of pyridoxal enzymes (Fig. 1).

Fig. 1: Homodimeric structure of D-THA DH and its clear
electron density for the cofactor PLP.

On the basis of the crystal structures of H351A
complexed with D-THA/L-EHA, a possible mechanism
for the catalytic reaction of D-THA DH could beproposed. The substrate covalently binds to the PLP
molecule in each active-site pocket of the homodimericstate of D-THA DH. The bound Mg2+ is also in direct
coordinated with the β-carboxyl and β-hydroxyl oxygen
atoms, presumably playing an important role in thestabilization of the bound substrate during the catalytic
reaction. After abstracting a proton from the α-carbon of

Photon Factory Activity Report 2014 #32 (2015) B

D-THA by Lys43 to generate a quinonoid resonancestructure, the β-elimination reaction occurs by a proton
donation to the substrate hydroxyl group. The
substrate/inhibitor complex structures also provided
important clues regarding substrate stereospecificity ofthe D-THA DH. The Cβ-hydroxyl group of the bound LEHA (substrate) to the poorly active H351A mutant
occupied a position close to the bound Mg2+ at the active
site, clearly indicating a possibility of metal-assisted Cβhydroxyl elimination from the substrate as describedabove (Fig. 2). In contrast, the Cβ-hydroxyl of the D-EHA(inhibitor) was bound far from the Mg2+ (Fig. 2). Theseobservations suggest that the substrate stereospecificity ofD-THA DH depends on the orientation of the Cβhydroxyl group at the active site, and its spatial
arrangement is compatible with the 3R configuration of 3hydroxyaspartate.

Fig. 2: Active-site snapshots for (A) the bound inhibitor
D-EHA to the wild-type enzyme and (B) the substrate L-EHA bound to the poorly active H351A mutant.

Acknowledgement
We are grateful to the beamline scientists at the PF(Tsukuba, Japan) for their kind assistance with all X-ray
diffraction experiments.
References[1] Maeda et al., J. Biochem. 148, 705–712 (2010).
[2] Matsumoto et al., Acta Crystallographica F69, 11311134 (2013).
[3] Matsumoto et al., Appl. Microbiol. Biotechnol., inpress. DOI 10.1007/s00253-015-6479-3 (2015).
* y-yasutake@aist.go.jp