Photon Factory Activity Report 2018 #36 (2019)

BL-5A, BL-17A/2017G033

Crystal structure of α-amylase II from Eisenia fetida

Yu Hirano1, Mitsuhiro Ueda2, Taro Tamada1,*1
National Institutes for Quantum and Radiological Science and Technology, Shirakata 2-4, Tokai,319-1106, Japan
2
Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka, 599-8531, Japan
1 Introduction
The earthworm Eisenia fetida has some cold-adapted
enzymes. We have reported two tertiary structures ofenzymes from E. fetida, endo-1,4-β-glucanase (Ef-EG2)[1] and endo-1,4-β-mannanase (Ef-Man) [2]. Here, wereport a raw-starch-digesting α-amylase from E. fetida (EfAmy II).
We have purified two α-amylases (Ef-Amy I and II),and they are composed of similar amino acids (89%sequence identity). However, the thermal stability andsubstrate specificity are different between Ef-Amy I and II.
Previously, we have determined the crystal structure of EfAmy I at 1.3 Å resolution. The overall structure of Ef-Amy
I comprises N-terminal TIM barrel and C-terminal β-barrel
domains. To understand the differences in the properties ofEf-Amy I and II, we have determined crystal structure of
Ef-Amy II.
2 Experiment
Crystallization experiments of Ef-Amy II wereperformed by the hanging-drop vapor-diffusion method at
293 K. Crystals were obtained with the reservoir solution
containing poly (acrylic acid sodium salt) 5100 as aprecipitant. Crystals were cooled in a nitrogen-gas stream
at 100 K during X-ray data collection.
Diffraction data sets were collected at the BL-17A
beamline. The wavelength of X-rays was set to 0.98 Å anddiffraction intensities were measured with Eiger X 16M
detector. The crystal structure of Ef-Amy II wasdetermined by the molecular replacement method with thecrystal structure of Ef-Amy I as a search model.
3 Results and Discussion
The diffraction data sets were obtained at 2.5 Åresolution (Table 1; the statistics for Ef-Amy I are shownas well). The crystal belongs to the space group P212121
with unit cell parameters of a = 95.5 Å, b = 100.5 Å, c =118.9 Å.
The crystal structure of Ef-Amy II consists of Ala17Val506, which includes all mature peptides (Gln18Val506). The Rwork and Rfree factors after structure refinement
were 17.8% and 23.6%, respectively. The overall structureof Ef-Amy II shows high similarities to that of Ef-Amy I,
and the superposition of the two structures indicates ther.m.s.d. of 0.64 Å for 484 Cα atoms (Fig. 1a).
The estimated catalytic residues D213, E249 and D316
show high structural similarities between Ef-Amy I and II(Fig. 1b). However, Ef-Amy II does not have the Gly-richloop, which exists in Ef-Amy I and is considered to affectsubstrate binding in human salivary α-amylase [3]. This
structural difference around the active site between Ef-Amy I and II seems to effect the substrate specificity. To
confirm the roles of this loop, we continue structuralstudies of α-amylases from E. fetida in combination withbiochemical studies.
Table 1: Diffraction data statistics
Resolution (Å)

Ef-Amy II

Ef-Amy I

50-2.50(2.60-2.50)

50-1.30(1.38-1.30)

Space group

P212121

P3221

Unit Cell a, b, c (Å)

95.5, 100.5, 118.9

96.2, 96.2, 122.0

Completeness (%)

99.9 (99.8)

99.8 (99.0)

Rsym (%)

12.7 (54.6)

5.6 (78.8)<I>/<σ(I)>14.6 (4.0)

15.1 (2.1)

Fig. 1 Superposition between Ef-Amy II (blue) and I(pink). (a) The overall structures. (b) The structures
around estimated active sites.
Acknowledgement
We thank the PF beamline staff for data collection.
References[1] T. Arimori et al., J. Synchrotron Rad. 20, 884 (2013).
[2] M. Ueda et al., Enzyme Microb. Technol. 117, 15 (2018).
[3] N. Ramasubbu et al., J. Mol. Biol. 325, 1061 (2003).
* tamada.taro@qst.go.jp