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J. Biol. Chem., Vol. 280, Issue 42, 35126-35135, October 21, 2005

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Active-site Peptide "Fingerprinting" of Glycosidases in Complex Mixtures by Mass Spectrometry

DISCOVERY OF A NOVEL RETAINING -1,4-GLYCANASE IN CELLULOMONAS FIMI^*

From the Protein Engineering Network of Centres of Excellence of Canada, Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada

New proteomics methods are required for targeting and identification of subsets of a proteome in an activity-based fashion. Here, we report the first gel-free, mass spectrometry-based strategy for mechanism-based profiling of retaining -endoglycosidases in complex proteomes. Using a biotinylated, cleavable 2-deoxy-2-fluoroxylobioside inactivator, we have isolated and identified the active-site peptides of target retaining -1,4-glycanases in systems of increasing complexity: pure enzymes, artificial proteomes, and the secreted proteome of the aerobic mesophilic soil bacterium Cellulomonas fimi. The active-site peptide of a new C. fimi -1,4-glycanase was identified in this manner, and the peptide sequence, which includes the catalytic nucleophile, is highly conserved among glycosidase family 10 members. The glycanase gene (GenBank^TM accession number DQ146941) was cloned using inverse PCR techniques, and the protein was found to comprise a catalytic domain that shares 70% sequence identity with those of xylanases from Streptomyces sp. and a family 2b carbohydrate-binding module. The new glycanase hydrolyzes natural and artificial xylo-configured substrates more efficiently than their cello-configured counterparts. It has a pH dependence very similar to that of known C. fimi retaining glycanases.

Received for publication, August 1, 2005

^* This work was supported by the Protein Engineering Network of Centres of Excellence of Canada, a Natural Sciences and Engineering Research Council of Canada postgraduate scholarship (to O. H.), a Michael Smith Foundation for Health Research postdoctoral fellowship (to Y.-W. K.), and a Killam postdoctoral fellowship (to S. J. W.). 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 nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) DQ146941.

¹ Both authors contributed equally to this work.

² Present address: School of Chemistry, University of Melbourne, Parkville, Victoria 3010, Australia.

³ To whom correspondence should be addressed: Dept. of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada. Tel.: 604-822-3402; Fax: 604-822-8869; E-mail: withers{at}chem.ubc.ca.

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