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

6A, 12NW/2004G355

Crystallographic analysis of maltohexaose-producing amylasefrom alkalophilic Bacillus sp.707
Ryuta KANAI1,2, Toshihiko AKIBA2, Keiko HAGA1, Kunio YAMANE1, and Kazuaki HARATA*,2
1
Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.
2
Biological Information Research Center, National Institute of Advanced Industrial Science andTechnology, Tsukuba, Ibaraki 305-8566, Japan.
Introduction
G6-amylase (E.C.3.2.1.98) from alkalophilic Bacillus
sp.707, which belongs to the glycoside hydrolase family
13, predominantly produces maltohexaose (G6) fromstarch and related α-1, 4-glucans. To elucidate thereaction mechanism of G6-amylase, crystal structures
were determined for the native enzyme and its complex
with pseudo-maltononaose [1].

resolution. On the electron density maps, the sugar
molecule was initially constructed as an α-1, 4-linked
chain of nine 6-deoxy-D-glucoses, and then the structure
was corrected to Acv-Glc-Glc-Glc-Acv-Glc-Glc (Acv,acarviosine, disaccharide analogue unit; Glc, glucose).
The final R and Rfree values were 17.2% and 20.7%,respectively. Atomic coordinates have been deposited
with Protein Data Bank (native structure: 1WP6, complex
structure: 1WPC).

Results and Discussion
Preparation and crystallization
G6-amylase from alkalophilic Bacillus sp.707 wasexpressed by Bacillus subtilis 207-25. The enzyme wascrystallized by the hanging drop vapor diffusion method
using the reservoir solution containing 50% (v/v) 2methylpentane-2, 4-diol, 100 mM Tris-HCl (pH 8.5) and200 mM ammonium phosphate. Calcium chloride andsodium chloride were added to final 1 mM in the dropsolution. Rod-like crystals were grown within 4 days atroom temperature. Crystals of the complex with pseudomaltononaose were obtained by soaking the crystal for 3
days in the crystallization solution containing 10 mM
acarbose and 10 mM maltotriose.

Measurement of X-ray diffraction data
X-ray diffraction measurements for the crystals ofnative G6-amylase and its pseudo-maltononaose complex
were carried out at the BL-6A and AR-NW12 station,
respectively. Both the crystals belong to the space group
P212121 and the unit cell dimensions are a = 47.6 Å, b =82.8 Å, and c = 127.2 Å for the native crystal and a =47.4 Å, b = 82.5 Å, and c = 126.9 Å for the complex.
Intensity data were collected at 100K to the resolution of
1.94 Å, and 32,772 and 39,475 unique reflections wereobtained with the completeness of 98.7% and 98.8%, andRmerge of 4.8% and 5.1% for the native and sugar complex
crystals, respectively.
Structure determination and refinement
The crystal structure of native G6-amyalse wasdetermined by molecular replacement and refined at 2.1
Å resolution. The final R and Rfree values were 16.6% and21.0%, respectively. The structure of the pseudomaltononaose complex was determined by the sameprocedure using the native structure and refined at 1.9 Å210

Users' Report

Description of the structure and discussion
G6-Amylase consists of three domains A, B and C.
Domain A (5 –105, 208 – 396) forms a (β/α)8 barrel likethe other enzymes of α-amylase family, domain B (106207) is attached on the domain A, and domain C consistsof β-strands. The backbone structure of G6-amylse isvery similar to those of some liquefying α-amylases withan average difference of less than 1.0 Å in their2+equivalent Cα position. G6-amylase contains the Ca +2+
Na -Ca metal ion triad at the interface between domain
A and domain B.
The crystal structures revealed that Asp236 is a
nucleophilic catalyst and Glu266 is a proton
donor/acceptor. The enzyme has three and six subsites atreducing- and non-reducing-end side, respectively. The
pseudo-maltononaose molecule was observed at thesubsites –6 to +3 in the active site cleft like an enzymesubstrate complex. The structure of the pseudomaltononaose, Acv-Glc-Glc-Glc-Acv-Glc-Glc, suggeststhat it is derived from acarboses and maltotrioses byintermolecular transglycosylation and hydrolysis. Thereare a few direct and water-mediated contacts between thesugar residues and subsites –2 and –3. The indole moietyof Trp140 is closely stacked on the cyclitol and 4-amino6-deoxyglucose residues located within 4 Å distance at
subsites –6 and –5, respectively. Such a face-to-face
short contact indicating strong hydrophobic interaction
may regulate the disposition of the glucose residue atsubsite –6 to dominantly produce maltohexaose.
References[1] R. Kanai et al., Biochemistry, 43, 14047 (2004).
* k-harata@aist.go.jp