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

18B, 12NW/2004G355

Crystallographic analysis of the sugar complexes of maltohexaose-producing
amylase from 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. Previously we havefound that Trp140 is a key residue for the G6 production.
To elucidate the role of Trp140, crystal structures of theG6-amylase complexes with G5 and G6 were determined
and compared with the structure of the previously
reported pseudo-maltononaose complex [1, 2].

complex was determined by the same procedure using thenative structure and refined at 1.9 Å resolution. The final
R and Rfree values were 15.5% and 18.4%, respectively.
The structures of G5 and G6 were constructed based on
3Fo-2Fc and Fo-Fc electron density. In addition, several
short sugar molecules were modeled on the molecularsurface in the both crystals. Atomic coordinates havebeen deposited with Protein Data Bank (G5 complex:2D3L, G6 complex: 2D3N).

Results and Discussion

Description of the structure and discussion
Our previous crystallographic study showed thatG6-amylase has nine subsites, from –6 to +3, and pointed
out the importance of the indole moiety of Trp140 in G6production. G6-amylase has very low levels of hydrolytic
activities for oligosaccharides shorter than maltoheptaose.
In the active site of the G6-amylase/G5 complex, G5 isbound to subsites –6 to –2 while G1 and G6 are found atsubsites +2 and –7 to –2, respectively, in the G6amylase/G6 complex. This indicates that such a binding
mode prevents G6 from being hydrolyzed to shorter
oligosaccharides. In both structures, the glucosyl residue
located at subsite –6 is stacked to the indole moiety of
Trp140 within a distance of 4 Å. The sugar molecules arealso stabilized by hydrogen bonds with several residues in
the active site cleft. In addition to the sugar molecules
bound to the active site, several sugar molecules arebound on the molecular surface; two G1 and one G2 inthe crystal of G5 complex, and two G2, one G3, and one
G4 in the crystal of G6 complex. The measurement of theactivities of the mutant enzymes when Trp140 wasreplaced by leucine (W140L) or by tyrosine (W140Y)
showed that the G6 production from short-chain amylose
by W140L is lower than that by W140Y or wild type
enzyme. The face-to-face short contact between Trp140
and substrate sugars is suggested to regulate thedisposition of the glucosyl residue at subsite –6 and togovern product specificity for G6 production.

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) and
200 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. The crystals of the complexes with G5and G6 were prepared by soaking for 30 minutes in asolution containing 40 mM G5 or 75 mM G6.

Measurement of X-ray diffraction data
X-ray diffraction measurements for the crystals of theG6-amylase complexes with G5 and G6 were carried out
at the BL-18B and AR-NW12 station, respectively. Boththe crystals belong to the space group P212121 and the unit
cell dimensions are a = 47.5 Å, b = 82.5 Å, and c = 126.9Å for the G5 complex and a = 47.6 Å, b = 82.7 Å, and c =127.0 Å for the G6 complex. Respective intensity data
were collected at 100 K to the resolution of 2.06 Å and1.94 Å, and 31,896 and 40,560 unique reflections wereobtained with the completeness of 97.8% and 98.7%, andRmerge of 16.8% and 4.8% for the complexes with G5 andG6, respectively.
Structure determination and refinement
The crystal structure of the G6-amyalse complex withG5 was determined by molecular replacement and refinedat 2.3 Å resolution. The final R and Rfree values were116.8% and 21.3%, respectively. The structure of the G6

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References[1] R. Kanai et al., Biochemistry, 43, 14047 (2004).
[2] R. Kanai et al., Protein Sci., 15, 468 (2006).
* k-harata@aist.go.jp