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J. Biol. Chem., Vol. 281, Issue 25, 17099-17107, June 23, 2006

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Family 6 Carbohydrate Binding Modules in -Agarases Display Exquisite Selectivity for the Non-reducing Termini of Agarose Chains^*

Joanna Henshaw, Ami Horne-Bitschy, Alicia Lammerts van Bueren, Victoria A. Money^¶, David N. Bolam, Mirjam Czjzek^||, Nathan A. Ekborg^**, Ronald M. Weiner^**, Steven W. Hutcheson^**, Gideon J. Davies^¶, Alisdair B. Boraston¹, and Harry J. Gilbert²

From the Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom, the Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada, the ^¶Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, United Kingdom, the ^||Station Biologique de Roscoff, Vegetaux Marins et Biomolecules, UMR7139-CNRS-UPMC, Place George Teissier, B. P. 74, 29682 Roscoff, France, and the ^**Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742

Carbohydrate recognition is central to the biological and industrial exploitation of plant structural polysaccharides. These insoluble polymers are recalcitrant to microbial degradation, and enzymes that catalyze this process generally contain non-catalytic carbohydrate binding modules (CBMs) that potentiate activity by increasing substrate binding. Agarose, a repeat of the disaccharide 3,6-anhydro--L-galactose-(1,3)--D-galactopyranose-(1,4), is the dominant matrix polysaccharide in marine algae, yet the role of CBMs in the hydrolysis of this important polymer has not previously been explored. Here we show that family 6 CBMs, present in two different -agarases, bind specifically to the non-reducing end of agarose chains, recognizing only the first repeat of the disaccharide. The crystal structure of one of these modules Aga16B-CBM6-2, in complex with neoagarohexaose, reveals the mechanism by which the protein displays exquisite specificity, targeting the equatorial O4 and the axial O3 of the anhydro-L-galactose. Targeting of the CBM6 to the non-reducing end of agarose chains may direct the appended catalytic modules to areas of the plant cell wall attacked by -agarases where the matrix polysaccharide is likely to be more amenable to further enzymic hydrolysis.

Received for publication, January 24, 2006 , and in revised form, April 3, 2006.

Note Added in Proof—The biochemical properties of Aga86E and Aga16B are now described in Ref. 61

^* This work was supported in part by Grant DEB0109869 from the National Science Foundation (to N. A. E., R. M. W., and S. W. H.), by a Biotechnology and Biological Sciences and Research Council project grant (to V. A. M., H. J. G., D. N. B., G. J. D., and J. H.), and by a grant from the Natural Sciences and Engineering Research Council of Canada (to A. B. B., A. H.-B., and A. L. v. B.). 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 supplemental Table 1S. The atomic coordinates and structure factors (code 2CDP and 2CDO) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

¹ A Canada Research Chair in Molecular Interactions.

² To whom correspondence should be addressed. Tel.: 44-0191-222-6962; Fax: 44-0191-222-7242; E-mail: H.J.Gilbert{at}ncl.ac.uk.

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