Crystal structure of Disproportionating Enzyme from Potato at 2.0 Å resolution

Kayo Imamura1, Takanori Matsuura2, Zhengmao Ye1, Takeshi Takaha3, Kazutoshi Fujii3, Masami Kusunoki2 and Yasunori Nitta1

1Graduate School of Agriculture and Biological Sciences, Osaka prefecture University,
2Institute for Protein Research, Osaka University,
3Biochemical Research Laboratory, Ezaki Glico Co., Ltd.

BSR2004 (the 8th International Conference on Biology and Synchrotron Radiation), (Himeji, Japan 2004.9.7-11)

Disproportionating enzyme (D-enzyme) is a member of the α-amylase family, whose core structure consists of a (α/β)8 barrel. A number of crystal structures of α-amylase family have been well investigated and from these structural studies, the presence of three domains emerged as a common structural feature, although additional domains are present in some enzyme. D-enzyme catalyzes intra-molecular transglycosylation (cyclization) , inter-molecular transglycosylation (coupling, disproportionation) and hydrolytic reaction of α-1,4-glucan. In contrast to the cyclodextrin glucanotransferase (CGTase), which synthesizes cyclodextrins with a ring size of 6-8 glucose units, D-enzyme produces those of more than 17 glucose units. To understand the difference in reaction specifisity of these two enzymes, we have determined two crystal structures of D-enzyme from potato with or without a substrate analog, acarbose, at 2.0 and 2.3 Å resolution, respectively. First structure is determined by multiple wavelength anomalous dispersion (MAD). D-enzyme is essentially composed of one domain with (α/β)8 barrel. Additionally, more than 20 amino acids from N-terminal are located in the other molecule which located in crystallographic symmetry, and have mainly hydrophobic interactions. This observation coincide D-enzyme from Potato is as a dimer in aqueous solution. The structure and function relationship of this enzyme will be discussed.

Table Data collection statistics
Beamline Photon Factory BL5A
Spacegroup C2221
Cell constant (Å) a=69.3, b=119.9, c=174.5
Wavelength (Å) 1.0000
Resolution range (Å) 50-2.31 (2.39-2.31)
Observed reflections 228119
Unique reflections 31950
Completeness (%) 99.0 (99.2)
<II> 2.6
Rmerge (%) 5.5 (16.2)
Structure of D-enzyme (dimer)

Fig 1. Structure of D-enzyme (dimer)

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