Photon Factory Activity Report 2022 #40 (2023)

BL-5A, BL-17A, AR-NW12A /2021G508, 2019G518

X-ray structure of a homodimeric L-ribulose 3-epimerase from Methylomonas sp.
Hiromi Yoshida1,2*, Akihide Yoshihara2,3, Shiro Kato2,3, Susumu Mochizuki2,3, Kazuya Akimitsu2,3,Ken Izumori2,3, and Shigehiro Kamitori1,2
1
Department of Basic Life Science, 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, 2393 Ikenobe, Miki, Kagawa, 761-0795, Japan.
1 Introduction
D-Allulose is a rare sugar, and it shows physiological
functions, such as suppressing postprandial blood glucose
elevation and suppressing fat accumulation. To date,
several enzymes capable of D-allulose production havebeen reported, and they belong to ketose 3-epimerase
family, including D-tagatose 3-epimerases (DTEs,homodimer),D-allulose
3-epimerases(DAEs,homotetramer) and L-ribulose 3-epimerases (LREs,homotetramer). Based on the sequence similarity with thepotential enzymes, a putative protein from Methylomonas
sp. DH-1 (NCBI, WP_064020855.1, 286 a.a.) was selected
as a putative sugar isomerase/epimerase. The recombinant
putative protein exhibited the highest enzymatic activity
towards L-ribulose. It was considered to be L-ribulose 3epimerase (LRE), but also had relatively high enzymatic
activity towards D-allulose. In this study, we determined
the X-ray structure of LRE from Methylomonas sp.
(MetLRE). The X-ray structure analysis of MetLRE
revealed a homodimer different from previously reported
homotetrameric LREs, and the most structurally similarprotein to MetLRE was D-allulose 3-epimerase fromArthrobacter globiformis (AgDAE), which has a long Cterminal -helix. The X-ray structure of MetLRE is thefirst known homodimeric L-ribulose 3-epimerase with ashort C-terminal -helix [1].
2 Experiment
The recombinant MetLRE was expressed using a
synthesized gene encoding the putative sugar
isomerase/epimerase from Methylomonas sp. DH-1 andused for crystallization. Crystals of C-terminal His-tagged
MetLRE were obtained in a droplet containing a mixtureof 0.8 l protein solution (5.5 mg/ml in 5 mM Tris-HCl,pH 8.0) and 0.8 l reservoir solution (0.2 M MgCl2, 0.1 MTris-HCl pH 8.5, 25 % (w/v) PEG3350) in a wellcontaining 50 l reservoir solution by the sitting-drop
vapor-diffusion method at 293 K. As for the crystals of Nterminal His-tagged MetLRE, protein solution (22.3mg/ml) in 5 mM Tris-HCl, pH 8.0 and the reservoirsolution (0.1 M HEPES pH 7.5, 10 % (w/v) polyethylene
glycol 8,000 and 8 % (v/v) ethylene glycol) were used withthe above crystallization conditions.
X-ray diffraction data were collected on the PF BL-5A
and the PF BL-17A in the KEK, and processed using theprograms HKL2000, XDS and the CCP4 suite. Thestructure was determined by molecular replacement withthe program MOLREP using the structure of AgDAE(PDB ID 5ZFS) .
3 Results and Discussion
MetLRE_his in complex with D-fructose wascrystallized in the orthorhombic space group P212121 withtwo molecules in an asymmetric unit. MetLRE forms ahomodimer (Mol-A and Mol-B) (Fig. 1). In Mol-A, the Cterminus was refined to His293 including a linker and partof the His-tag, and the C-terminus of Mol-B was refined toGly287, which is the beginning of a linker before the Histag.

Mol-AMol-BNNCCFig. 1: Dimeric structure of MetLRE in complex withD-fructose. The bound metal ions are shown in bluespheres. N and C indicate the N terminus and Cterminus, respectively.
C7Mol-A

6
8N
b1 b8b2

5b6Mn0
1b7

6’b3b4b5

4

8’
2
3'3Fig. 2: Monomeric structure of MetLRE.

Photon Factory Activity Report 2022 #40 (2023)

Fig. 3: Overall structure of the structurally similar enzymes.
The monomeric structure (up) and overall structure (down) of structurally similar enzymes arerepresented. (a) A. globiformis ketose 3-epimerase (AgDAE), (b) M. loti L-ribulose 3-epimerase(MlLRE), (c) P. cichorii D-tagatose 3-epimerase (PcDTE), and (d) A. tumefaciens D-allulose 3epimerase (AtDAE). The C-terminal -helix is labeled as 8.

The monomeric structure has twelve -helices and eightb-strands, forming a (b)8 barrel fold with four additional
short -helices (0, 3’, 6’ and 8’) (Fig. 2). There is ametal ion at the center of the barrel, which is coordinatedby Glu152, Asp185, His211 and Glu246 to form thecatalytic site and bind the substrate.
The most structurally similar protein was ketose 3epimerase from Arthrobacter globiformis (previouslyknown as AgDAE, L-ribulose 3-epimerase, PDB code5zfs), with 54 % identity, 1.6 Å r.m.s.d. and 45.6 Z-scoreby Dali search. Other similar proteins were L-ribulose 3epimerase from Mesorhizobium loti (MlLRE, 3vyl, 40 %,1.3 Å, 42.5), D-tagatose 3-epimerase from Pseudomonas
cichorii (PcDTE, 2qul, 30 %, 1.7 Å, 37.3) and D-allulose
3-epimerase from Agrobacterium tumefaciens (AtDAE,2hk1, 28 %, 1.8 Å, 36.8). The monomeric structures ofthese proteins are compared in Fig. 3. The structures adopt(b)8 barrel fold, and a metal ion is bound at the center ofthe each barrel. Although the features of LREs are a long
C-terminal helix (8, 26-29 residues) and formation of
stable homotetramers, MetLRE has a shorter C-terminal
helix (8, 16 residues) than previously reported LREs andforms a homodimer like homodimeric PcDTE. A similarfeature of MetLRE with LRE was the angle of the catalytic
metal – N-terminus of 8 – C-terminus of 8 (metal-8N8C), which was 54 º, being between MlLRE (58 º) andAgDAE (52 º), and different from PcDTE (49 º) andAtDAE (43 º) (Fig. 3).

In this study, a novel homodimeric L-ribulose 3epimerase from Methylomonas sp. was expressed and its
X-ray structure was determined. The structure showed aunique short C-terminal -helix compared to the known
structures of the structurally similar homotetrameric Lribulose 3-epimerases and D-allulose 3-epimerases.
Acknowledgement
We thank the PF staff for the support of data collection.
References[1] Yoshida, H. et al., FEBS Open Bio. 11, 1621-1637(2021).
* yoshida.hiromi@kagawa-u.ac.jp