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J. Biochem, 2003, Vol. 134, No. 6 881-891
© 2003 Japanese Biochemical Society

BIOCHEMISTRY

Effects of Essential Carbohydrate/Aromatic Stacking Interaction with Tyr100 and Phe259 on Substrate Binding of Cyclodextrin Glycosyltransferase from Alkalophilic Bacillus sp. 1011

Keiko Haga*,1, Ryuta Kanai*,1,2, Osamu Sakamoto1, Masanobu Aoyagi1, Kazuaki Harata§,2 and Kunio Yamane,1

1 Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572; and 2 Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566

The stacking interaction between a tyrosine residue and the sugar ring at the catalytic subsite –1 is strictly conserved in the glycoside hydrolase family 13 enzymes. Replacing Tyr100 with leucine in cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. 1011 to prevent stacking significantly decreased all CGTase activities. The adjacent stacking interaction with both Phe183 and Phe259 onto the sugar ring at subsite +2 is essentially conserved among CGTases. F183L/F259L mutant CGTase affects donor substrate binding and/or acceptor binding during transglycosylation [Nakamura et al. (1994) Biochemistry 33, 9929–9936]. To elucidate the precise role of carbohydrate/aromatic stacking interaction at subsites –1 and +2 on the substrate binding of CGTases, we analyzed the X-ray structures of wild-type (2.0 Å resolution), and Y100L (2.2 Å resolution) and F183L/F259L mutant (1.9 Å resolution) CGTases complexed with the inhibitor, acarbose. The refined structures revealed that acarbose molecules bound to the Y100L mutant moved from the active center toward the side chain of Tyr195, and the hydrogen bonding and hydrophobic interaction between acarbose and subsites significantly diminished. The position of pseudo-tetrasaccharide binding in the F183L/F259L mutant was closer to the non-reducing end, and the torsion angles of glycosidic linkages at subsites –1 to +1 on molecule 1 and subsites –2 to –1 on molecule 2 significantly changed compared with that of each molecule of wild-type-acarbose complex to adopt the structural change of subsite +2. These structural and biochemical data suggest that substrate binding in the active site of CGTase is critically affected by the carbohydrate/aromatic stacking interaction with Tyr100 at the catalytic subsite –1 and that this effect is likely a result of cooperation between Tyr100 and Phe259 through stacking interaction with substrate at subsite +2.

* These authors contributed equally to this work.

§ Atomic coordinates for the complex have been deposited with the Brookhaven Protein Data Bank (file names: wild-type-acarbose, 1UKQ; F183L/F259L-acarbose, 1UKS; Y100L-acarbose, 1UKT).

To whom correspondence should be addressed. Tel & Fax: +81-29-853-6680, E-mail address: kyamane{at}sakura.cc.tsukuba.ac.jp


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