Photon Factory Activity Report 2014 #32 (2015) B

BL-5A, BL-17A, AR-NW12A, AR-NE3A/2013G506

X-ray structure of Cellulomonas parahominis L-ribose isomerase
Yuji Terami2, Hiromi Yoshida1*, Keiko Uechi2, Kenji Morimoto2, Goro Takata2, Shigehiro Kamitori1
1Life Science Research Center and Faculty of Medicine, Kagawa University, Miki-cho, Kita-gun,Kagawa 761-0793, Japan
2
Rare Sugar Research Center, Kagawa University, Miki-cho, Kita-gun, Kagawa 761-0795, Japan
1 Introduction
L-Ribose is non-natural sugar, so-called rare sugar, andis not generally used in metabolic pathway as a carbon
source. An enzyme, L-ribose isomerase (L-RI) fromAcinetobacter sp. strain DL28 (AcL-RI) was reported as anew enzyme which can constitutively produce L-ribose
isomerase [1], and had no significant sequence similarity
to known protein structures [2, 3], implying an uniquestructure to recognize L-ribose as its ideal substrate. In
previous study, we have determined X-ray structure ofAcL-RI [4], and here we determined X-ray structure of LRI from Cellulomonas parahominis MB426 (CpL-RI)
that has a broader substrate specificity and more thermal
stability than AcL-RI [5,6].

salt bridge with Lys93. It was expected that these charged
residues were sequentially aligned on β-sheet 1 torecognize the hydroxyl groups of the substrate.

2 Experiment
The expression and purification of CpL-RI werereported [5]. Crystals of his-tagged CpL-RI were grownin a droplet mixing 2 µl of protein solution (30 mg ml-1 in
5 mM Tris-HCl, pH 8.0) and 2 µl of reservoir solution(3.9 M ammonium acetate, 0.1 M sodium acetate
trihydrate pH 4.6) against 450 µl of the reservoir solution
at 293 K. X-ray diffraction data were collected on theBL5A in the PF. Diffraction data were processed using
the programs HKL2000 and the CCP4 program suite. The
structure of CpL-RI was solved by molecular replacement
using the structure of AcL-RI.

(b)

3 Results and Discussion
The subunit structure of CpL-RI was very similar to thatof AcL-RI. CpL-RI adopted a cupin-type β-barrel
structure, having four α-helices (H1, H3, H4, and H7),
three 310-helices (H2, H5, and H6), and two large β-sheets(β-sheet 1 and β-sheet 2) formed by 11 β-strands (B1–B11) (Fig. 1a). Two molecules in the asymmetric unit
(Mol-A and Mol-B) formed a homo-dimer with 2-foldsymmetry, and this dimer formed a homo-tetramer withthe symmetry-operated dimer (Mol-C and Mol-D) (Fig.
1b). The β-sheet 1 formed by six β-strands (B1, B2, B3,B10, B5, and B8) was located at the interface between
Mol-A and Mol-B, contributing to the intermolecularinteractions of the dimer.
In the structure of the catalytic site of CpL-RI, a boundmetal ion was coordinated by His106, His108, Glu113,His188, and two waters (W1 and W2), giving anoctahedral form of metal coordination (Fig. 1c). A large
space for a substrate was identified between β-sheets 1and β-sheet 2. Glu113 formed hydrogen bonds with W1and W2, and Lys111 formed a hydrogen bond with W1.
Glu204 directed its side chain to the catalytic center by aAcknowledgement
We thank the PF staff for the support of data collection.
This research was performed with the approval of thePhoton Factory Advisory Committee and the National
Laboratory for High Energy Physics (2011G504 and2013G506), Japan.

(a)

(c)

Fig. 1: The structure of CpL-RI
The subunit structure (a),tetramer structure (b), and thecatalytic site structure of CpLRI (c) are shown. Selectedamino acid residues involved in
the dimer-dimer interactions areindicated by orange sticks (a).

References[1] T. Shimonishi & K. Izumori. J. Ferment. Bioeng. 81(1996) 493-497.
[2] R.M. Mizanur et al., Biochim Biophys Acta. 1521
(2001) 141-145[3] H. Yoshida et al., Acta Crystallogr. F67 (2011) 12811284.
[4] H. Yoshida et al., FEBS J. 281 (2014) 3150-3164.
[5] K. Morimoto et al., J Biosci Bioeng. 115 (2013) 377–381.
[6] Y. Terami et al., Appl Microbiol Biotechnol. 2015 Feb
8. [Epub ahead of print].
* h.yoshi@med.kagawa-u.ac.jp