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J. Biol. Chem., Vol. 279, Issue 4, 3014-3024, January 23, 2004

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Crystal Structures of Geobacillus stearothermophilus -Glucuronidase Complexed with Its Substrate and Products

MECHANISTIC IMPLICATIONS^*

Gali Golan, Dalia Shallom¶||, Anna Teplitsky, Galia Zaide¶, Smadar Shulami¶, Timor Baasov**, Vivian Stojanoff, Andy Thompson, Yuval Shoham¶||, and Gil Shoham¶¶

From the Department of Inorganic Chemistry and the Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel, the ^¶Department of Food Engineering and Biotechnology, ^||Institute of Catalysis Science and Technology, and ^**Department of Chemistry, Technion-ITT, Haifa 32000, Israel, and European Synchrotron Radiation Facility, Avenue des Martyrs, Grenoble 38043, France

-Glucuronidases cleave the -1,2-glycosidic bond between 4-O-methyl-D-glucuronic acid and short xylooligomers as part of the hemicellulose degradation system. To date, all of the -glucuronidases are classified as family 67 glycosidases, which catalyze the hydrolysis via the investing mechanism. Here we describe several high resolution crystal structures of the -glucuronidase (AguA) from Geobacillus stearothermophilus, in complex with its substrate and products. In the complex of AguA with the intact substrate, the 4-O-methyl-D-glucuronic acid sugar ring is distorted into a half-chair conformation, which is closer to the planar conformation required for the oxocarbenium ion-like transition state structure. In the active site, a water molecule is coordinated between two carboxylic acids, in an appropriate position to act as a nucleophile. From the structural data it is likely that two carboxylic acids, Asp³⁶⁴ and Glu³⁹², activate together the nucleophilic water molecule. The loop carrying the catalytic general acid Glu²⁸⁵ cannot be resolved in some of the structures but could be visualized in its "open" and "closed" (catalytic) conformations in other structures. The protonated state of Glu²⁸⁵ is presumably stabilized by its proximity to the negative charge of the substrate, representing a new variation of substrate-assisted catalysis mechanism.

Received for publication, September 11, 2003 , and in revised form, October 9, 2003.

The atomic coordinates and structure factors (codes 1K9D (WT AguA), 1K9E (E285N-MeGlcA complex), 1K9F (E285N-aldotetraouronic acid complex), 1L8N (WT-products complex), and 1MQR (E386Q)) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This study was supported by grants from the Israel Science Foundation (to G. S. and Y. S.), by the French-Israeli Association for Scientific and Technological Research (AFIRST) (to Y. S.) (Jerusalem, Israel) and by the German-Israeli Foundation for Scientific Research and Development (to Y. S., G. S., and T. B.). Additional support was provided by the Otto Meyerhof Center for Biotechnology, established by the Minerva Foundation (Munich, Germany). 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.

The on-line version of this article (available at http://www.jbc.org) contains an additional figure.

These two authors contributed equally to this work and were supported by the Wolf Foundation.

To whom correspondence may be addressed. Tel.: 972-4-8293072; Fax: 972-4-8293399; E-mail: yshoham{at}tx.technion.ac.il. ¶¶ To whom correspondence may be addressed. Tel.: 972-2-6585611; Fax: 972-2-6585319; E-mail: gil2{at}vms.huji.ac.il.

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