Photon Factory Activity Report 2019 #37 (2020)

AR-NE3A, BL-5A/2019G518

X-ray structure of Arthrobacter globiformis M30 ketose 3-epimerase
Hiromi Yoshida1,2*, Akihide Yoshihara2,3, Pushpa Kiran Gullapalli4, Kouhei
Ohtani4, Kazuya Akimitsu2,3, Ken Izumori2,3 and Shigehiro Kamitori1,21Life Science Research Center and Faculty of Medicine, Kagawa University, Kagawa, Japan
2
International Institute of Rare Sugar Research and Education, Kagawa University, Kagawa, Japan
3
Faculty of Agriculture, Kagawa University, Kagawa, Japan
4
Matsutani Chemical Industry Co. Ltd, Hyogo, Japan1 Introduction
The ketose 3-epimerase from Arthrobacter globiformis
M30 has been reported to be a D-allulose 3-epimerase(AgDAE), since its substrate specificity was similar tothose of the known D-psicose 3-epimerases from
Clostridium cellulolyticum (CcDPE) and Agrobacterium
fabrum (A. tumefaciens; AtDPE) [1]. D-Allulose (or Dpsicose) is a rare sugar, and its physiological functions(such as moderating blood glucose levels and fat
accumulation) are being focused on for human healthcare.
Therefore, the enzymes capable of D-allulose production
are attractive. We determined here, the X-ray structure ofAgDAE and found the structure is more similar to that ofL-ribulose 3-epimerase from Mesorhizobium loti (MlLRE)
than previously known structure of DPEs [2]. In addition,
we found that AgDAE shows the highest enzymatic
activity toward L-ribulose. Although AgDAE is considered
to be L-ribulose 3-epimerase, it is useful for D-allulose
production in the food industry, considering its enzymatic
activity toward D-allulose, thermal stability and wellknown microorganism recognized as safe.
2 Experiment
The recombinant AgDAE was used for crystallization.
Crystals were obtained in a in a droplet consisting of 1.0 lprotein solution (10 mg/ml in 5 mM Tris–HCl buffer pH8.0) and 1.0 l reservoir solution (0.1 M sodium acetate pH
4.6, 3.0–4.0 M ammonium acetate) against 50 l reservoir
solution by the sitting-drop method at 293 K. X-ray
diffraction data were collected on the PF-AR NE3A in theKEK, and processed using the programs HKL-2000 and
the CCP4 suite. Initial phase was determined by molecularreplacement using the structure of MlLRE (PDB code:3vyl) as a search model.
3 Results and Discussion
The structure of AgDAE was refined at a resolution of1.96 Å. Two molecules (Mol A and B) in an asymmetricunit (hexagonal space group P6522) formed ahomotetramer by a twofold symmetry operation (Fig.1).
The monomer structure adopted a ()8 barrel fold withthe catalytic residues and metal ion located in the center ofthe barrel, as is observed in the structures of sugarphosphate isomerases and epimerases (Fig. 2a).
The monomer structure and the overall structure of
AgDAE was highly similar to that of the homotetramericL-ribulose 3-epimerase from Mesorhizobium loti (Figs. 2aMol B

Mol A

Mol B’

Mol A’

Fig. 1: The overall structure of AgDAE.

(a)
7
8

6

8 7 6’
6
1Mn
5
0 2 3 4
1

C-terminal tail(b)

Mol A

8
5

4

8’
2

3'

3Ml-RE (3vyl)

AgDAE(5zfs)

(c)

(d)
8

8

CcDPE (3vnk)

AtDPE (2hk1)

Fig. 2: The monomer structure comparisons among related
enzymes. (a) A. globiformis D-allulose 3-epimerase(AgDAE), (b) M. loti L-ribulose 3-epimerase (MlLRE), (c)C. cellulolyticum D-psicose 3-epimerase (CcDPE), (d) A.
tumefaciens D-psicose 3-epimerase (AtDPE).

Photon Factory Activity Report 2019 #37 (2020)

and 2b). The common feature is in the lengths of their Cterminal helices (8). The helices (8) of AgDAE(Asp264–His289) and MlLRE (Asp265–Ala293) werelonger than those of CcDPE (Asp268–Glu288) and AtDPE(Asp268–Gly288). The longer helices extend along thetetramerization interface and probably stabilize thetetrameric structure. According to the structure of MlLRE
reported by Uechi et al., the longer C-terminal helix ofMlLRE with an additional C-terminal tail (Arg294–Pro297) promoted the formation of additional
intermolecular interactions and strengthened the stabilityof the enzyme compared with CcDPE and AtDPE [3].
Furthermore, we found that AgDAE showed a higher
enzymatic activity towards L-ribulose, followed by Dallulose (63.5% of L-ribulose). AgDAE could becategorized as an L-ribulose 3-epimerase but showed adifferent substrate specificity to MlLRE that is specific for
L-ribulose, followed by L-xylulose (42% of L-ribulose),
D-tagatose (24% of L-ribulose) and D-ribulose (20% of Lribulose) [4]. Therefore, AgDAE is useful enzyme for Dallulose production.
Acknowledgement
We thank the PF staff for the support of data collection.
References[1] Yoshihara, A. et al., J. Biosci. Bioeng. 123, 170-176(2017).
[2] Yoshida, H. et al., Acta Cryst. F74, 669-676 (2018).
[3] Uechi, K. et al., Acta Cryst. D69, 2330-2339 (2013).
[4] Uechi, K. et al., Biosci. Biotechnol. Biochem. 77, 511515 (2013).
* h.yoshi@med.kagawa-u.ac.jp