Photon Factory Activity Report 2016 #34 (2017)

BL-5A, NW12A, NE3A/2015G534

X-ray structures of the Pseudomonas cichorii D-Tagatose 3-epimerase mutant C66S
recognizing deoxy sugars as substrates
Hiromi Yoshida1*, Akihide Yoshihara2, Tomohiko Ishii3 Ken Izumori2 and Shigehiro Kamitori1
1Life Science Research Center and Faculty of Medicine, Kagawa University, Kagawa, Japan
2
Rare Sugar Research Center and Faculty of Agriculture, Kagawa University, Kagawa, Japan
3
Faculty of Engineering, Kagawa University, Hayashi-cho, Takamatsu, Kagawa, Japan
1 Introduction
D-Tagatose 3-epimerase (DTE) catalyzes epimerization
between D-tagatose and D-sorbose (Fig.1 (a)), and DTE
family enzymes are widely found in various
microorganisms. DTE from Pseudomonas cichorii(PcDTE) has a broad substrate specificity and catalyzes
the epimerization of not only D-tagatose to D-sorbose, butalso D-fructose to D-allulose that is one of rare sugars.
Therefore, PcDTE is used to produce D-allulose from themore abundant sugar D-fructose. We have alreadydetermined the X-ray structures of PcDTE in complexes
with D-tagatose and D-fructose, and proposed catalytic
reaction mechanism via a cis-enediolate intermediate [1].
In these X-ray structures, the hydroxyl groups (O1 andO3) and ketone group (O2) of the substrate were
important to be recognized by the enzyme with specificinteractions, while the interactions at the 4-, 5-, 6positions of substrate seemed to be recognized weakly by
the enzyme. In recent years, we found PcDTE can also
recognize deoxy sugars as ligand, such as 1-deoxy D-, Ltagatose, 6-deoxy L-allulose, and 1-deoxy 3-keto Dgalactitol (D3G) (Fig.1(c)). The last ligand, D3G has nohydroxyl group at 1- and 3- positions and no ketone
group at 2- position.

(a)

(b)

D-tagatose

D-sorboseD-fructose

D-allulose(c)6-deoxy
L-allulose

1-deoxy 3-keto
1-deoxy
D-galactitol (D3G) D-tagatose

1-deoxy
L-tagatose

Fig. 1: Chemical reaction catalyzed by PcDTE andligand molecules. (a, b) The epimerization reaction ofsubstrate. (c) The chemical structure of deoxy sugars.

In this study, we report the X-ray structure of PcDTE
mutant Cys66Ser (PcDTE C66S) in complexes with D3G,1-deoxy D-tagatose, and 1-deoxy L-tagatose, and show theunique recognition mode of PcDTE to deoxy sugars.
Since crystals of PcDTE C66S diffracted to a higher
resolution than wild-type PcDTE crystals without
significant differences in their structures, we used themutant enzyme.
2 Experiment
PcDTE C66S was expressed as His-tagged protein andpurified by affinity chromatography [2]. The purified
protein solution was concentrated to 6 - 7 mg/ml for
crystallization. Crystals were obtained in a droplet
containing 2.0 μ L of protein solution (6 - 7 mg/mL in 5
mM of Tris-HCl, pH 8.0) and 2.0 μL of reservoir solution
6.0 – 11.0 % (w/v) PEG 4000 and 100 mM CH3COONa,pH 4.6) against 450 μ L of the reservoir solution by thehanging-drop method at 293 K.
In addition, a crystal was grown in microgravity byutilizing the crystallization facilities of the JAPANAerospace Exploration Agency (JAXA) on board theJapanese Experiment Module Kibo in the International
Space Station (ISS) under a high-quality protein crystal
growth project (JAXA-PCG). In order to obtain crystals
grown in a microgravity environment in the ISS,crystallization was established using a counter-diffusion
method in solution containing 15 % (w/v) PEG 4000 and
100 mM CH3COONa, pH 4.6.
For obtaining a ligand-complexed structure, a single
crystal was soaked in 25 – 50 % (w/v) ligand molecules
in reservoir solution. X-ray diffraction data were collectedon the PF BL-5A PF-AR NW12A, NE3A, and a Rigaku
R-AXIS VII imaging system on a Rigaku RA-Micro7HF
rotating anode (CuK) X-ray generator with ValiMax
optics (40 kV, 30 mA). Diffraction data were processedusing the programs HKL2000, the CrystalClear system
and CCP4 program suite.
Initial phase determination of PcDTE C66S wasperformed by molecular replacement using the structureof PcDTE (PDB ID:2QUL) as a probe model.
3 Results and Discussion
The overall structure of PcDTE C66S was equivalent
to the previously reported structure of wild-type PcDTE.

Photon Factory Activity Report 2016 #34 (2017)(1) 1-Deoxy D-tagatose and 1-deoxy L-tagatose
The electron density of the bound 1-deoxy D-tagatose
at the catalytic site indicated that the bound substrate
adopts two alternative conformations, Form A and Form
B, as shown in Fig. 2(a, b). In Form A, O2 and O3
coordinate to the metal ion, and O5 and O6 interact with
Ser66 directly or via a water molecule. In Form B, thedirection of the bound substrate molecule is opposite tothat of Form A, in which O6 interacts with Glu158,His188, and Arg217. O5 and O4 coordinate to the metalion. O2 forms a water-mediated hydrogen bond with
Ser66 (Cys66 in wild-type PcDTE). The ketone group ofthe substrate is required to coordinate to the metal ion forthe epimerization reaction. The binding mode of Form A
is suitable for the epimerization reaction, while that ofForm B cannot lead to the epimerization reaction, whichis assumed to be the inhibitor-binding mode. The
molecule of 1-deoxy L-tagatose is found to bind to thecatalytic site in the inhibitor-binding mode (Fig. 2(c)), as
found in Form B of 1-deoxy D-tagatose in which O6
interacts with Glu158, His188, and Arg217, O5 and O4
coordinate to the metal ion, and O2 forms a hydrogenbond with Glu152.

substrate to the proper orientation. This may be the reason
why PcDTE efficiently catalyzes the epimerization ofvarious ketoses including deoxy sugars as substrates.

(a)

(b)
Asp185

His188Glu246
Asp185Glu246
His188

Arg217

Arg217

His211

23 4

Glu158

His211564

156

Glu152

1

Glu152
Trp113

Leu108Ser66

Glu15832

Trp113
Leu108Ser66

1-deoxy D-tagatose (Form A)

1-deoxy D-tagatose (Form B)

(c)

(d)

Glu246
His188
Asp185
Arg217

Asp185Glu246
His188
Arg217

His211

Phe248(2) 1-Deoxy 3-keto-D-galactitol (D3G)
The catalytic site structure with the bound 1-deoxy 3keto-D-galactitol (D3G) is shown in Fig. 2(d). O3 (ketonegroup) and O4 (hydroxyl group) of D3G coordinate to themetal ion, forming hydrogen bonds with His188 andGlu152. Glu246 directs its OE2 atom to the hydrogen
atom attached to C4 with a distance of 2.3 Å. O2 forms
hydrogen bonds with Glu158, His188, and Arg217. C1(methyl group) is located in the hydrophobic pocket
formed by Trp113, Phe248, and Val259. O5 does notform a hydrogen bond with the enzyme, and O6 interacts
with Ser66 by a water-mediated hydrogen bond. Thisstructure shows that the epimerization reaction occurs at
the 4-position of D3G to give 1-deoxy 3-keto D-allitol asa product. The positon of a ketone group coordinating to
the metal ion (O3 of D3G or O2 of D-tagatose) is strictly
fixed by interactions between the hydroxyl group (O2 ofD3G or O1 of D-tagatose) and enzyme.
PcDTE catalyzes the epimerization of ketohexoses atthe 3-position adjacent to the ketone group at the 2position (Fig. 1 (a, b)). The ketone group (O2) andhydroxyl group at the 3-position (O3) are required tocoordinate to the metal ion at the catalytic site of theenzyme for the epimerization reaction, and this correct
metal coordination by the substrate is performed by therecognition of a hydroxyl group at the 1-position (O1)with Glu158, His188, and Arg217. Thus, PcDTE strictlyrecognizes the substrate at the 1-, 2-, and 3-positions. On
the other hand, PcDTE loosely recognizes the substrate at
the 4-, 5-, and 6-positions with a few specific interactions
between the substrate and enzyme. The substrate
recognition at the 1-, 2-, and 3-positions is responsible forenzymatic activity, and substrate-enzyme interactions atthe 4-, 5-, and 6-positions are not essential for thecatalytic reaction of the enzyme; they merely adjust

5His211

6Glu152

42

Glu158Glu15231
Leu108

4 32
56
Leu108

Trp113
Ser66

1Glu158 Val259
Trp113

Ser66
Ser37

Trp15
Ser37
Trp15

1-deoxy L-tagatose (microgravity)

1-deoxy 3-keto D-galactitol (D3G)

Fig. 2: Structure of the catalytic site of PcDTE C66S.
Acknowledgement
We thank the members of JAXA for assisting withcrystallizations under microgravity and also thank the PF
staff for the support of data collection. This research wasperformed with the approval of the Photon Factory
Advisory Committee. This study is contributed by a partof “High-Quality Protein Crystal Growth Experiment on
KIBO” promoted by JAXA (Japan Aerospace
Exploration Agency). Russian Space craft “Progress”
or/and “Soyuz” provided by Russian Federal Space
Agency were used for space transportation. A part ofspace crystallization technology had been developed by
European Space Agency and University of Granada.
References[1] Yoshida et al., J. Mol. Biol., 374(2), 443-453, (2007).
[2] Yoshida et al., Appl Microbiol Biotechnol. 100(24),10403-10415, (2016).
* h.yoshi@med.kagawa-u.ac.jp