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J. Biol. Chem., Vol. 282, Issue 23, 17242-17249, June 8, 2007

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Three-way Stabilization of the Covalent Intermediate in Amylomaltase, an -Amylase-like Transglycosylase^*

Thomas R. M. Barends¹, Jelle B. Bultema, Thijs Kaper^¶, Marc J. E. C. van der Maarel^¶, Lubbert Dijkhuizen^¶, and Bauke W. Dijkstra²

From the Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, Centre for Carbohydrate Bioprocessing, TNO-University of Groningen, Kerklaan 30, 9751 Haren, and ^¶Laboratory of Microbiology, University of Groningen, Kerklaan 30, 9751 Haren, The Netherlands

Amylomaltases are glycosyl hydrolases belonging to glycoside hydrolase family 77 that are capable of the synthesis of large cyclic glucans and the disproportionation of oligosaccharides. Using protein crystallography, we have generated a flip book movie of the amylomaltase catalytic cycle in atomic detail. The structures include a covalent glycosyl enzyme intermediate and a covalent intermediate in complex with an analogue of a co-substrate and show how the structures of both enzyme and substrate respond to the changes required by the catalytic cycle as it proceeds. Notably, the catalytic nucleophile changes conformation dramatically during the reaction. Also, Gln-256 on the 250s loop is involved in orienting the substrate in the +1 site. The absence of a suitable base in the covalent intermediate structure explains the low hydrolysis activity.

Received for publication, February 19, 2007 , and in revised form, March 27, 2007.

The atomic coordinates and structure factors (code 2OWW, 2OWC, and 2OWX) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by European Union 5FP CEGLYC Project (Contract QLK3-CT-2001-00149). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Max Planck Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany.

² To whom correspondence should be addressed. Fax: 31-50-3634800; E-mail: b.w.dijkstra{at}rug.nl.

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