Photon Factory Activity Report 2017 #35 (2018)

Crystal structure of α-amylase from Eisenia fetida

BL-17A/2017G033

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
We have already reported that the earthworm Eisenia
fetida has some cold-adapted enzymes, and furthermore,
we have reported two tertiary structures of enzymes fromE. fetida, endo-1,4-β-glucanase (Ef-EG2) [1] and endo1,4-β-mannanase (Ef-Man) [2]. Here, we report a novel
raw-starch-digesting α-amylase from E. fetida (Ef-Amy I).
We have purified two α-amylases (Ef-Amy I and II),
which are electrophoretically homogeneous states. EfAmy I and II are composed of similar amino acids (89%sequence identity), however, the cold adaptability and thestarch-digesting activity are different between Ef-Amy Iand II. To understand those differences at atomic level,
we have determined crystal structure of Ef-Amy I.
2 Experiment
Ef-Amy I crystals were obtained under the conditions
containing lithium sulphate (crystal-1), PEG1000(crystal-2), ammonium citrate (crystal-3), or PEG3350(crystal-4) as precipitants. 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-Amy I weredetermined by the molecular replacement method withthe crystal structure of porcine pancreatic α-amylase(PDB 1KXQ) [3] as a search model.
3 Results and Discussion
Prismatic crystals were obtained in everycrystallization condition. The diffraction data sets of all
crystals were obtained higher than 1.9 Å resolution (Table1), especially crystal-1 and 4 were diffracted to 1.3 Å and1.4 Å resolutions, respectively. All crystals belong to thespace group P3221 with unit cell parameters of a = b =96~97 Å, c = 121~122 Å.
The final model of Ef-Amy I (crystal-1) consists ofAla17-Ala513, which includes all mature peptides(Gln18-Val510). The final Rwork and Rfree (5%) factors were
14.7% and 16.8%, respectively. Overall structure of EfAmy I comprises a TIM barrel, which is the common foldin GH13 family proteins, and C-terminal domain(including Greek key motif) (Fig. 1a), and shows highsimilarities to the crystal structures of α-amylase fromother species. Superposition of the Ef-Amy I structure tothe amylase structures shows rms distances of 0.89 Å for
450 Cα atoms (Porcine pancreatic, 1PIG) [4], 0.87 Å for

450 Cα atoms (Human pancreatic, 4W93) [5], and 0.91 Åfor 429 Cα atoms (Tenebrio molitor, 1JAE) [6].
The molecular surface of Ef-Amy I is negatively
charged as well as that of Ef-EG2 and Ef-Man (Fig. 1b).
In the crystal-1 structure of Ef-Amy I, one acetate ion that
included in the crystallization solution was bound to theactive site. To obtain the structure of Ef-Amy II, we
continue crystallization experiment.
Table 1: Diffraction data statistics
crystal-1

crystal-2

crystal-3

crystal-450-1.30(1.38-1.30)

50-1.90(2.02-1.90)

50-1.70(1.81-1.70)

50-1.40(1.49-1.40)

96.2, 122.0

97.1, 121.4

96.1, 121.1

96.4, 121.2

99.8 (99.0)

99.9 (99.7)

99.8 (98.7)

99.9 (99.4)

Rsym (%)

5.1 (67.3)

8.1 (55.8)

7.0 (46.9)

6.2 (49.6)<I>/<σ(I)>15.1 (2.1)

12.3 (2.3)

14.0 (3.4)

14.1 (2.7)

Resolution (Å)
Unit Cell a, c(Å)
Completeness(%)

Fig. 1: Crystal structure of Ef-Amy I.

(a) Ribbon diagram. (b) Electrostatic potentials.

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] A. Desmyter et al., J. Biol. Chem. 277, 23645 (2002).
[4] M. Machius et al., J. Mol. Biol. 260, 409 (1996).
[5] L. K. Williams et al., Nat. Chem. Biol. 11, 691 (2015).
[6] S. Strobi et al., J. Mol. Biol. 278, 617 (1998).
* tamada.taro@qst.go.jp