Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

NW12/2005G286

Crystal structure of β-1,3-xylanase catalytic module (XYL4-CM)
from Vibrio sp. AX-4 in an atomic resolution
Keishi Sakaguchi1, Takashi Kawamura1, Masashi Kiyohara2,Nobuhisa Watanabe*1, Min Yao 1, Kuniko Yamaguchi2, Makoto Ito2, Isao Tanaka1
1
Hokkaido Univ., Sapporo 060-0810, Japan1
Kyushu Univ., Fukuoka 812-8581, Japan
Introduction
β-1,3-Xylanase (EC 3.2.1.32) is an enzyme capable ofhydrolyzing the β-1,3-xylosidic linkages of β-1,3-xylan toproduce β-1,3-xylooligosaccharides with a different
xylose unit. β-1,3-xylanase from Vibrio sp. AX-4, termed
XYL4, is a modular enzyme composed of a catalyticmodule (XYL4-CM) belonging to glycoside family 26(GH-26) and tandem xylan-binding mocules (XYL4XBMs) [1].
Recently, we reported that the crystallization andpreliminary X-ray analysis of XYL4-CM [2]. The crystalof XYL4-CM belonged to space group P212121, with unitcell parameters a=51.6, b=75.8, c=82.0 Å. X-ray
diffraction data was collected to 1.44 Å resolution [2].
Here, we report the atomic resolution X-ray crystal
structure of XYL4-CM. This is the first analysis of β-1,3xylanase. Investigating XYL4-CM structure andcomparing it with structures of other GH-26 enzymes
would be facilitate the structural and functional studies of
GH-26 and β-1,3-xylanase.
Experimental Procedures
As described elsewhere, Se-Met crystals of XYL4-CM
was obtained by hanging drop vapor diffusion method at
293 K, and the structure determination of XYL4-CM wascarried out by multiple-anomalous diffraction method.
Furthermore, 0.900 Å wavelength was selected to Xray measurement under 100 K on the PF NW12 station ofSpring-8. Data set was integrated with HKL2000 [3], andscaled with SCALEPACK Structure of XYL4-CM wasdetermined using the molecular replacement method.
Manual model fitting using the program XtalView/Xfit[4], and the structure refinement was carried out using
REFMAC [5]. The stereochemical quality of the final
refined models was analyzed by the program
PROCHECK [6]. Final coordinates and the structure
factors of XYL4-CM were deposited in the protein Data
Bank with code 2DDX.
Results
The crystal structure of XYL4-CM was determined at
an atomic resolution of 0.86 Å. The final model of
XYL4-CM exhibited an R-factor of 0.140 (R-free=0.148)
includes 324 out of 355 residues with no break. However,
the C- and N-terminal regions of the model, 1-22 and344-355 respectively, could not be built due to poor
electron density.

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Users' Report

The overall fold of XYL4-CM revealed a classic (β/α)8barrel (TIM-barrel), the same as other members of GH-26.
The two glutamate residues of Glu-116 and Glu-212,
which were previously demonstrated as an acid/basecatalyst and a nucleophile by sequence and mutation
analyses [1], located close to the C-termini of the forthand seventh β strands, respectively. These features of
XYL4-CM are typical for enzymes classified in clan GHA (also called the 4/7 superfamily), which includes GH26.
A DALI [5] search revealed that the three dimensional
structure of XYL4-CM shows a significant similarity withthose of other GH-26 enzymes as follows; β-mannanase
A from Cellvibrio japonicus (CjMan26A, Z score 19.7
with a root mean square deviation [rmsd] of 3.5 Å), βmannanase from Cellulomonas fimi (CfMan26A-50K, Z
score 17.0 with a rmsd of 3.4 Å), lichenase fromClostridium thermocellum (CtLic26A, Z score of 16.7
with a rmsd of 3.3 Å).
To characterize function of the active site region inmore detail, crystallization and structure determination ofXYL4-CM in complex with the substrate (β-1,3xylooligosacchrides) has been conducted extensively.
References[1] M. Kiyohara et al., Biochem. J. 388, 949 (2005).
[2] K. Sakaguchi et al., Acta Crystallogr. D Biol.
Crystallogr. 60, 1470 (2004).
[3] Z. Otwinowski et al., Meth. Enzymol. 276, 307 (1997)[4] D. E. McRee, J. Struct. Biol. 125, 156 (1997)[5] G. N. Murshudov et al., Acta Crystallogr. D Biol.
Crystallogr. 53, 240 (1997)[6] R. A. Laskowski et al., J. Appl. Crystallogr. 26, 283(1993)*nobuhisa@sci.hokudai.ac.jp