Photon Factory Activity Report 2014 #32 (2015) B
BL-1A, BL-5A, BL-17A, AR-NE3A, AR-NW12A/2010G596, 2012G748

Crystal structure of glycine oxidase from Geobacillus kaustophilus
Takako Shiono1, Takaomi Nomura1,§, Yoshiaki Nishiya2, and Ryoichi Arai1,*1Fac. of Tex. Sci. Tech., Shinshu Univ., Ueda, Nagano 386-8567, Japan
2
Fac. of Sci. Tech., Setsunan Univ., Neyagawa, Osaka 572-8508, Japan
1 Introduction
Glycine oxidase (Gox; EC 1.4.3.19) catalyzes theoxidative deamination of small amines and D-amino acids.
This enzyme has potential for industrial applications. We
investigated Goxs derived from several gram-positive
Bacillus species. However, those specific activities andstabilities were lower than industrial requirements. In thepresent study, we targeted a Gox from Geobacillus
kaustophilus (GoxGk). GoxGk has high thermal stability,
but it is subject to substrate inhibition. To elucidate thecharacteristic properties of GoxGk, the three-dimensional
structural information is needed. Here we report thecrystal structure of GoxGk.
2 Experiments
GoxGk was expressed in E. coli, and purified by thefollowing steps: ammonium sulfate fractionation,hydrophobicchromatography,
anion
exchange
chromatography, and gel filtration chromatography. The
purified enzyme showed a single band in SDS-PAGE.
GoxGk was crystallized at 20ºC using the hanging drop
vapor diffusion method. 1 μl of GoxGk was mixed withthe same volume of reservoir solution (7 % (v/v) 2propanol, 0.1 M phosphate-citrate pH 4.7, 0.2 M LiSO4).
X-ray diffraction data collections were performed at KEK
PhotonFactory
macromolecular
crystallography
beamlines at 95 K with reservoir solution added to 20
mM glycine as a substrate and 28 % (v/v) 2-propanol as acryoprotectant. The structure was solved by molecularreplacement method using Phaser with model structuresof Gox from Bacillus subtilis (GoxBs; PDB code, 1RYI)[1]. The crystal structure was refined using COOT andREFMAC5.
3 Results and Discussion
The GoxGk crystal belongs to the hexagonal spacegroup P6522, with unit cell constants of a = 87.99 Å, b =152.29 Å, c = 413.44 Å, and contains two protein
molecules per asymmetric unit. The structure was refined
to 2.2 Å resolution (PDB code 4YSH; Rwork = 23.3%, Rfree= 26.6%). The crystal structure and gel filtration
chromatography analysis show that GoxGk forms
tetramer in the same way as GoxBs. Each GoxGk
monomer comprises eight α-helices, four 310 helix, andsixteen β-strands, and contains one noncovalently-bound
FAD molecule. The FAD-binding domain has theconserved Rossmann fold βαβ motif, which serves as adinucleotide-binding motif. In the two monomer
molecules of GoxGk per asymmetric unit, the substrate,
glycine, bound to one molecule and did not bind toanother molecule. The monomeric structures of GoxGkand GoxBs overlap well (r.m.s.d. = 1.2–1.3 Å). Analysis
by Protein Interactions Calculator (PIC) [2] shows that
their differences in types of intersubunit interactions:
there are more hydrophobic and ionic interactions inintersubunit of GoxGk than those of GoxBs.
In the active site pockets of GoxGk and GoxBs, theconformations of several residues are highly conserved.
The substrate-binding residues, Tyr246 and Arg302 ofGoxBs, correspond to Tyr253 and Arg309 of GoxGk,suggesting their common mechanism of enzymaticreaction.
The present study provides new structural insights ofGoxGk. To understand the characteristic properties ofGoxGk in detail, further analysis is currently in progress.

Fig. 1: Crystal structure of the GoxGk tetramer.
Acknowledgements
We thank the PF staff for help of X-ray diffraction
experiment. This work was supported by Grants-in-Aidfor Scientific Research (KAKENHI) Grant Numbers
22113508, 24113707, and 24780097, funded by MEXT
and, JSPS Japan.
References[1] M. Mörtl et al., J. Biol. Chem. 279, 29718 (2004).
[2] K. G. Tina et al., Nucleic Acids Res. 35, W473 (2007).
§*

nomurat@shinshu-u.ac.jp
rarai@shinshu-u.ac.jp