Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science

NW12, 5A/2004G372

X-ray Crystallographic analysis of heterotetrameric sarcosine oxidase
Koh Ida1*, Tomotaka Moriguchi2, and Haruo Suzuki1,2
Department of Biosciences, School of Science, and 2Division of Biosciences, Graduate School ofFundamental Life Science, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555,
Japan
1Introduction
The concentration of serum creatinine is widely used as
an indicator of renal function. Sarcosine oxidase (SO)[sarcosine: oxygen oxidoreductase (demethylating), EC
1.5.3.1] has been used in the enzymatic determination ofcreatinine in the clinical laboratory. SO has been isolated
from various bacterial strains. It was first purified from
Corynebacterium sp. U-96 and shown to be aheterotetrameric enzyme (Mr 174,000). The enzyme iscomposed of four nonidentical subunits (α, Mr 110,000; β,Mr 44,000; γ, Mr 21,000; δ, Mr 10,000). To understandthe structure-function relationship of SO more in detail,we aimed to determine the three dimensional structure ofthe enzyme.
Experiments and Result
Sarcosine oxidase was expressed and purified aspreviously described [1], except that the chromatographic
procedures were performed by ÄKTA prime system(Amersham Bisciences), using HiTrap DEAE FF, HisTrap
HP, HiTrap Phenyl FF, Resources Q and HiLoad 16/60
Superdex 200 prep grade columns (AmershamBiosciences). The purifed SO in 10 mM Tris-HCl (pH8.0) was concentrated to approx. 5 mg/ml. Crystallization
was performed by vapour diffusion method. Flower likecrystals were obtained by ammonium sulfate as aprecipitant. Refinement of the crystallization conditions
to 0.1 M Tris-HCl (pH 8.5), 1.9 M ammonium sulfate,and 10 mM CuSO4 improved the quality and size of thecrystals (Figure 1).
Diffraction data for HgSO4 derivative data sets andnative data set were collected on NW12 in PF-AR using
an ADSC Quantum 210 CCD detector. A crystal of theprotein was frozen using a cryoprotectant solution
containing 30% (v/v) glycerol in the crystallization
mother liquor. The data were processed using theMOSFLM (CCP4 suite) and scaled using the SCALA(CCP4 suite). The data processing statistic are given Table
1. After measurement, the crystal lost yellow colour. The
crystals have two problems that the cell dimensions are
too large and the crystal take large radiation damage.
These causes overlap and non-isomorphism. To obtainhigh quality data, we used large detector or fine slice
method and minimum X-ray exposed time. Structure
determination by the multiple-wavelength anomalous
diffraction (MAD) method is in progress.

Figure 1. Crystal of sarcosine oxidase

Table 1. Data processing statistics
Data set

NativeS. G.

P6522

Celldimensions

a=199.1
c=197.2

a=199.4
c=197.3

Resolution

2.152.85

WavelengthPeak1.00868

Edge

1.00934

Remote1.02000

Oscillation

0.3

0.5

Observ. ref.

1276782114744511506361153212Unique ref.

124207

53641

53807

53934

Comp.

99.8

100

100100Rmerge

9.2

8.9

9.1

9.4

I/σ

5.1

7.0

7.0

6.6

Multiplicity

10.3

21.4

21.4

21.4

References[1] H. Suzuki et al., Biosci. Biotechnol. Biochem. 69 (5),952-956 (2005).
*idakoh@sci.kitasato-u.ac.jp

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