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J Am Chem Soc. 2008 Jan 9;130(1):327-35. Epub 2007 Dec 8.

Synthesis and use of mechanism-based protein-profiling probes for retaining beta-D-glucosaminidases facilitate identification of Pseudomonas aeruginosa NagZ.

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Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, B.C. Canada.


The NagZ class of retaining exo-glucosaminidases play a critical role in peptidoglycan recycling in Gram-negative bacteria and the induction of resistance to beta-lactams. Here we describe the concise synthesis of 2-azidoacetyl-2-deoxy-5-fluoro-beta-d-glucopyranosyl fluoride as an activity-based proteomics probe for profiling these exo-glycosidases. This active-site directed reagent covalently inactivates this class of retaining N-acetylglucosaminidases with exquisite selectivity by stabilizing the glycosyl-enzyme intermediate. Inactivated Vibrio cholerae NagZ can be elaborated with biotin or a FLAG-peptide epitope using the Staudinger ligation or the Sharpless-Meldal click reaction and detected at nanogram levels. This ABPP enabled the profiling of the Pseudomonas aeruginosa proteome and identification at endogenous levels of a tagged protein with properties consistent with those of PA3005. Cloning of the gene encoding this hypothetical protein and biochemical characterization enabled unambiguous assignment of this hypothetical protein as a NagZ. The identification and cloning of this NagZ may facilitate the development of strategies to circumvent resistance to beta-lactams in this human pathogen. As well, this general strategy, involving such 5-fluoro inactivators, may prove to be of general use for profiling proteomes and identifying glycoside hydrolases of medical importance or having desirable properties for biotechnology.

[Indexed for MEDLINE]

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