Photon Factory Activity Report 2014 #32 (2015) B

BL-5A/2014G702

Engineering SS bridge of Thermostable Mutant of Bacillus sp. TB-90 Urate Oxidase
Takao Hibi1,*, Yuta Hayashi1, Takafumi Itoh1, and Yoshiaki Nishiya2
1
Fukui Prefectural University, Fukui 910-1195, Japan
2
Setsunan University, Neyagawa, Osaka 572-8508, Japan
1 Introduction
Urate oxidase from Bacillus sp. TB-90 (BTUO), one of
the most thermostable urate oxidases, is secreted as atetrameric protein of identical subunits.[1, 2] We
previously reported that binding of a sulfate anion to theenzyme induced the thermal stabilization, because thebound sulfate formed a salt bridge with two Arg298s inthe symmetrical related subunits.[3] To extensively
characterize the sulfate-binding site at the subunit
interface, site-directed mutagenesis of Arg298 to Cys wasdone, although the distance between the Cβ atoms ofArg298s seemed to be too long to introduce anengineering disulfide bond. However, this substitution
markedly increased the protein melting temperature of theR298C mutant by ~20°C compared with the wild-typeone, and then the thermostabilization was canceled by thereduction with dithiothreitol. Here we show the crystal
structure of R298C at 2.05 Å resolution, and demonstratethat the symmetrical related Cys298 residues are linked
with each other via a disulfide bridge.
2 Experiment
Crystals of the R298C mutant enzyme were grown by
the hanging-drop vapour-diffusion technique aspreviously described.[3] A protein solution (5 ml) wasmixed with an equal volume of reservoir solution
containing 16%(w/v) polyethylene glycol 8000, 100 mM
Tris-HCl pH 8.0, 0.08 M Li2SO4 and 1 mM 9-methyluric
acid. The crystal was momentarily soaked in the reservoir
solution containing 20% polyethylene glycol 400 and wasflash-cooled in a 100 K dry nitrogen stream and thenexposed to 1 Å X-ray beam. A native data set wascollected to 2.05 Å resolution using an ADSC Quantum
315 CCD camera and synchrotron radiation on beam line
BL-5A (Photon Factory, Tsukuba, Japan). Individualframes consisted of a 0.5° oscillation angle measured for
10 sec. Intensity data were processed, merged, and scaledwith HKL2000. Data collection statistics are given inTable 1.
Table 1: Data Collection and Refinement Statistics
data collection	  space group
P21212	  unit cell parameters (Å) a = 131.86, b = 142.58, c = 70.65	  resolution range (Å)
50 - 2.05 (2.09 - 2.05)	  total reflections
410,560	  no. of unique reflections
83,842	  completeness (%)
99.8 (99.9)
	  Rmerge(=ΣhΣi|Ih,i - <Ih>| / ΣhΣi|Ih,i|)0.121 (0.715)
	  I/σ20.3 (3.0)
	  redundancy
4.9 (4.8)

refinement	  refinement resolution (Å)
	  Rwork/Rfree	  RMSD	  bond length (Å)
	  bond angle (°)
	  Ramachandran plot	  favored region	  allowed region

32.27 - 2.05
0.169 / 0.221
0.0070.839
98.1%
1.9%

The structure of R298C mutant has been solved bymolecular replacement techniques using the program
PHENIX. The initial phase was solved using the model
3WLV as a search probe. The MR solution was readily
obtained and was rebuilt and refined. After several roundsof refinement, the electron density from the omit map
depicted a clear density for disulfide bridges. Statistics forrefinement are also given in Table 1.
3 Results and Discussion
The crystal structure of R298C was determined incomplex with the competitive inhibitor 9-methy uric acidat 2.05 Å resolution. Superposition of the R298C
structure onto that of the wild-type enzyme using GASH
revealed a rmsd of 0.28 Å for 1187 Cα atoms. Although
the differences of overall structures were negligible, theelectron densities around residues Pro287-Glu290 werecompletely lost in the middle of interfce loop II of R298Cmutant.
A clear electron density for a left-handed disulfide
bridge at the subunit interface was depicted from the σAomit map. The disulfide bond formation shortened thedistance between the Cβ atoms of Cys298s to be 4.54 Å,
0.9 Å shorter than that of Arg298s of the wild-type
enzyme. Instead, the averaged Cβ-Sγ-Sγ angle of 126.4°
was abnormally large, and the averaged χ2 torsion angle(Cα-Cβ-Sγ-Sγ) was 5.5°, so that a steric hindrance arose
between the Cα-Cβ and the Sγ-Sγ bonds. Further studies
on the thermal stabilization is in progress.
Acknowledgement
We thank the staff at the Photon Factory (Tsukuba,Japan) for providing data collection facilities and support.
References[1] Yamamoto, K., et al., J. Biochem. 119, 80-84 (1996).
[2] Nishiya, Y. et al., J. Anal. Bio-Sci. 23, 443-446 (2000).
[3] Hibi, T. et al. Biochemistry, 53 (24): 3879-3888(2014).
* hibi@fpu.ac.jp