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J. Biol. Chem., Vol. 278, Issue 48, 47394-47399, November 28, 2003

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Identification of the Catalytic Nucleophile of the Family 29 -L-Fucosidase from Thermotoga maritima through Trapping of a Covalent Glycosyl-Enzyme Intermediate and Mutagenesis^*

Chris A. Tarling, Shouming He, Gerlind Sulzenbacher¶, Christophe Bignon¶, Yves Bourne¶, Bernard Henrissat¶, and Stephen G. Withers||

From the Department of Chemistry, University of British Columbia, Vancouver V6T 1Z1, Canada and ^¶Architecture et Fonction des Macromolécules Biologiques, UMR 6098, CNRS and Universités Aix-Marseille I & II, 31 Chemin Joseph Aiguier, F-13402 Marseille Cedex 20, France

Fucose-containing glycoconjugates are key antigenic determinants in many biological processes. A change in expression levels of the enzymes responsible for tailoring these glycoconjugates has been associated with many pathological conditions and it is therefore surprising that little information is known regarding the mechanism of action of these important catabolic enzymes. Thermotoga maritima, a thermophilic bacterium, produces a wide range of carbohydrate-processing enzymes including a 52-kDa -L-fucosidase that has 38% sequence identity and 56% similarity to human fucosidases. The catalytic nucleophile of this enzyme was identified to be Asp-224 within the peptide sequence ²²²WNDMGWPEKGKEDL²³⁵ using the mechanism-based covalent inactivator 2-deoxy-2-fluoro--L-fucosyl fluoride. The 10⁴-fold lower activity (k_cat/K_m) of the site-directed mutant D224A, and the subsequent rescue of activity upon addition of exogenous nucleophiles, conclusively confirms this assignment. This article presents the first direct identification of the catalytic nucleophile of an -L-fucosidase, a key step in the understanding of these important enzymes.

Received for publication, June 23, 2003 , and in revised form, September 14, 2003.

Note Added in Proof—During the review process, a paper appeared describing the identification of the equivalent residue as the nucleophile in another family 29 -L-fucosidase (39).

^* This work was supported in part by the Protein Engineering Network of Centres of Excellence of Canada and the Natural Sciences and Engineering Research Council of Canada. 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.

Supported by a Post-doctoral Scholarship from the Royal Society, United Kingdom.

^|| To whom correspondence should be addressed. Tel.: 604-822-3402; Fax: 604-822-8869; E-mail: withers{at}chem.ubc.ca.

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