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

5A, 6A/2005G047

Crystal structure of Aspergillus niger ATCC9642 isopullulanase
Takashi TONOZUKA* 1, Masahiro MIZUNO1, Akihiro YAMAMURA1, Atsushi KOIDE1, Hiromi
YOSHIDA2, Shigehiro KAMITORI2, Atsushi NISHIKAWA1, Yoshiyuki SAKANO1
1

Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8
Saiwai-cho, Fuchu, Tokyo 183-8509, Japan2
Molecular Structure Research Group, Information Technology Center, Kagawa University, 1750-1
Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, JapanIntroductionEnzymes hydrolyze pullulan are useful in theproduction of various oligosaccharides. Isopullulanase(IPU) from Aspergillus niger ATCC9642 was originallyreported to be an enzyme which hydrolyzes specific sitesof pullulan to produce isopanose (Glc-α-(1→4)-Glc-α(1→6)-Glc). Although IPU does not hydrolyze starch, theenzyme hydrolyzes substrates containing panose (Glc-α(1→6)-Glc-α-(1→4)-Glc) motif, which is also found in
the structure of starch. Due to the unique properties, IPU
has been assigned its own EC number (EC 3.2.1.57).
Except for IPU, pullulan-hydrolyzing enzymes, such as
pullulanases, Thermoactinomyces vulgaris α-amylases,
and neopullulanases, are classified into glycosylhydrolase family (GH) 13, known as the α-amylase
family. In contrast, IPU is the sole enzyme which isclassified into GH 49 among these pullulan-hydrolases,
and no homology between IPU and α-amylase family
enzymes has been found. Interestingly, IPU does nothydrolyze dextran at all, while all other GH 49 enzymes
are dextran-hydrolyzing enzymes, such as endodextranase and isomaltotrio-dextranase. In this report, wedescribe the crystal structure of IPU.
Materials and Methods
Since the active IPU was not obtained fromrecombinant E. coli cells, Pichia pastoris was used for theheterologous expression of IPU [1]. The purification ofIPU was performed using a hydrophobic column. IPU
produced in P. pastoris was highly glycosylated. In manycases, glycosylation is an obstacle of crystallization, thus
thepurifiedIPU
wasdeglycosylatedusing
endoglycosidase Hf, and further purified using an ionexchange column [2]. Crystals of IPU were grown at 20°C using the hanging drop vapor-diffusion method, with awell solution of 20 % (w/v) PEG 8000 in 100 mM sodium
acetate buffer (pH 4.6) and a protein solution of 14 mg/ml.
The diffraction data was collected under cryogenic
condition, and the structure was solved by molecularreplacement using Penicillium minioluteum dextranase(1OGM) as the search model.
Results and Discussion
The asymmetric unit contains two molecules of IPU.
The structure of IPU is composed of N-terminal and C-218

Users' Reportterminal parts, which are designated domains N and C(Fig. 1). Domain N is composed of a β-sandwich.
Domain C has a right-handed parallel β-helical fold.
Three β-sheets form a coil, and the β-helix contains 10coils. The amino acid sequence of IPU has 15 potential
glycosylation sites, and the electron densities of Nacetylglucosamine residues were seen for the 11 sites.
The overall structures of IPU and P. minioluteum
dextranase resemble each other, while the shapes of theircatalytic clefts are different.

Domain C

Domain NFig. 1. Structure of IPU. N-acetylglucosamine residues
are indicated.
References[1] H. Akeboshi et al., Biosci. Biotechnol. Biochem. 67,1149 (2003).
[2] H. Akeboshi et al., Eur. J. Biochem. 271, 4420 (2004).
* tonozuka @ cc.tuat.ac.jp