Photon Factory Activity Report 2018 #36 (2019)

BL-5A/2017G610

X-ray structure of Oryza sativa hexokinase 6 in complex with -D-glucose

Hiromi Yoshida1,2*, Kazushi Matsudaira3, Susumu Mochizuki2,3 Ken Izumori2,3,Kazuya Akimitsu2,3and 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
3Faculty of Agriculture, Kagawa University, Kagawa, Japan
1 Introduction
Some hexokinases are found in Oryza sativa, and one ofwhich, hexokinase 6 (OsHXK6) shows enzymatic activity
not only to D-glucose but also to D-allose. D-Allose is
known as one of rare sugars which exist in a small amountin nature. Gibberellin is a plant hormone that is involvedin growth regulation. So far, D-allose is reported to inhibit
the gibberellin-dependent responses such as elongation ofrice leaves, and induce resistance to a phytopathogenic
bacterium Xanthomonas oryzae pv. oryzae including
induction of lesion mimic formation and pathogenesisrelated protein gene expression in rice [1-4]. D-Allose
suppresses expressions of gibberellin-responsive genes
through hexokinase-dependent pathway.
In this study, we determined crystal structure ofOsHXK6 in complex with -D-glucose and AMPPNP(ATP analogue) at 2.84 Å resolution.
2 Experiment
The recombinant OsHXK6 was used for crystallization.
After initial screening and optimization using theconcentrated protein solution (4.3 mg/mL), crystals wereobtained by mixing the protein solution (0.8 µL) and thesame volume of reservoir solution (0.1 M MOPSO, 0.1 M
Bis-Tris pH 6.5, 10 mM Spermine tetrahydrochloride, 10mM Spermidine trihydrochloride, 10 mM 1,4diaminodutane dihydrochloride, 10 mM D,L-Ornithine
monohydrochloride, 20% (w/v) 1,5- pentanediol, 10%(w/v) PEG8000) including 5 mM D-glucose, AMPPNP,
MgCl2, against 50 L of the reservoir solution by thesitting-drop method at 293 K. X-ray diffraction data werecollected on the PF BL-5A in the KEK, and processedusing the programs XDS and the CCP4 program suite.
Initial phase was determined by molecular replacement
using the structure of hexokinase 1 from Arabidopsis
thaliana (AtHXK1, PDB code: 4QS7) as a probe model.
3 Results and Discussion
OsHXK6 was crystallized in space group P3221, (a = b= 131.60 Å, c = 188.92 Å) and the structure was refined toR-factor of 0.206 at 2.84 Å resolution. The overall
structure of OsHXK6 with bound -D-glucose, AMPPNPand Mg is similar to that of AtHXK1 in -D-glucose boundform (4QS7, closed form) with 68 % identity, 1.2 Å rmsd,
human glucokinase in complex with -D-glucose and asmall molecule activator (4IXC, 35 %, 1.9 Å), hexokinasefrom Schistosoma mansoni complexed with -D-glucose(1BDG, 39 %, 1.8 Å) and AtHXK1 in ligand free form
(4QS8, open form, 67 %, 3.2 Å).

Fig. 1 shows the structure of OsHXK6 in complex with
-D-glucose, AMPPNP and Mg, and the active site
structure with bound -D-glucose. It showed the closed
form by the approach of the region colored in pale yellow(His102-Val236) including Thr202 and Lys203, to bind D-glucose.

AMPPNP

Lys203

Mg2+

Asp23865Thr202

1432

Glu323

Asn237
Asn264Glu292

Fig. 1: The structure of OsHXK6 in complex with -Dglucose, AMPPNP and Mg (deposited as 5ZQT, closedform), and the active site structure with bound -D-glucose.
Acknowledgement
We thank the PF staff for the support of data collection.
References[1] Kano, A. et al., Phytopathology. 100, 85-90 (2010).
[2] Fukumoto, T. et al., Planta. 234, 1083-1095 (2011).
[3] Kano, A. et al., J Exp Bot. 64, 4939-4951 (2013).
[4] Fukumoto, T. et al., Planta. 237, 1379-1391 (2013).
* h.yoshi@med.kagawa-u.ac.jp