Photon Factory Activity Report 2016 #34 (2017)

BL-17A/2015G325

Crystal structure of 1,4-β-mannanase from Eisenia fetida
Yu Hirano1, Mitsuhiro Ueda2, Taro Tamada1,*
1
National Institutes for Quantum and Radiological Science and Technology, Shirakata 2-4, Tokai,319-1116, Japan
2
Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka, 599-8531, Japan
1 Introduction
Endo-1,4-β-mannanase have been used for various
bioprocesses such as bleaching of softwood pulps,
declining viscosity of feeds and foods, and clarifying
beverages. 1,4-β-mannanase has been isolated frombacteria, fungi, higher plants, mollusks, and arthropods,
but it has not been reported about mannanase fromannelida earthworm.
We have found a high mannanase activity in the bodywall extract of the earthworm Eisenia fetida. We havecloned and expressed 1,4-β-mannanase from Eisenia
fetida (Ef-Man). To understand the structure and functionof Ef-Man, we have determined crystal structures of EfMan.
2 Experiment
Ef-Man crystals were obtained under the conditions
containing ammonium citrate (crystal-1) or sodium
potassium phosphate (crystal-2) as precipitants. Crystals(crystal-2) were soaked in 10 mM mannotriose (Man3)
for 1 day. The crystal-1 and crystal-2 were transferred tothe cryo-protectant solution consisting of the motherliquor with 20% glycerol. The crystals were cooled in anitrogen-gas stream at 100 K during X-ray data collection.
Diffraction data sets were collected on the BL-17A
beamline. The wavelength of X-rays was set to 0.98 Åand diffraction intensities were measured with Pilatus3
S6M detector. Crystal structures of Ef-Man weredetermined by the molecular replacement method withthe crystal structure of 1,4-β-mannanase from Mytilus
edulis (PDB 2C0H) [1] as a search model.
3 Results and Discussion
Thin plate crystals were obtained in both crystallization
conditions. The crystal-1 and crystal-2 were diffracted to1.7 Å and 1.6 Å resolutions, respectively (Table 1). Bothcrystals belong to the space group P212121 with unit cellparameters of a = 50.1 Å, b = 69.5 Å, c = 86.6 Å (crystal1) and a = 50.0 Å, b = 69.0 Å, c = 85.5 Å (crystal-2).
The final models of Ef-Man consist of Gln17-Gln377.
The overall structures of crystal-1 and crystal-2 showhigh similarities each other. The superposition of the twostructures indicated the root-mean-square distance of 0.18
Å for all Cα atoms. The crystal structure of Ef-Man also
shows high similarities to the crystal structures of 1,4-βmannanase from other species. Superposition of the EfMan structure to the mannanase structures shows rms
distances of 1.04 Å for 347 Cα atoms (M. edulis, 2C0H),
0.87 Å for 357 Cα atoms (Cryptococcus antaricticus,4OOU) [2], and 0.90 Å for 329 Cα atoms (Aplysiakurodai, 3VUP) [3].
In the crystal-1 structure of Ef-Man, one Tris moleculethat included in the crystallization solution was bound tothe active site. Although crystal-2 was soaked in theMan3 solution, there are no clear density of Man3 at theactive site of the crystal-2 structure. Instead of Man3,
water molecules were bound to the active site (Fig. 1). To
obtain the structure complexed with Man3, we will
modify soaking condition or perform co-crystallization
with substrates.
Table 1: Diffraction data statisticscrystal-2
crystal-1
BL-17A
Beamline
BL-17A
Wavelength (Å)
0.98
0.98
50-1.7050-1.60
Resolution (Å)(1.81-1.70)(1.70-1.60)
Unique reflections
62 97674 549Completeness (%)
98.4 (97.1)
99.4 (98.6)
Rsym (%)
7.4 (46.4)
9.7 (52.7)
13.1 (2.7)
10.3 (3.0)<I>/<σ(I)>Fig. 1: Electron density at the active site of Ef-Man.
2Fo-Fc map (blue mesh) of (a) crystal-1, (b) crystal-2.
Acknowledgement
We thank the PF beamline staff for data collection.
References[1] A.M. Larsson et al., JMB 357, 1500 (2006).
[2] M.K. Kim et al., Proteins 82, 3217 (2014).
[3] K. Mizutani et al., Acta Cryst. F 68, 1164 (2012).
* tamada.taro@qst.go.jp