| Synthesis and use of mechanism-based protein-profiling probes for retaining beta-D-glucosaminidases facilitate identification of Pseudomonas aeruginosa NagZ. | |
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MedLine Citation:
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PMID: 18067297 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Keith A Stubbs; Adrian Scaffidi; Aleksandra W Debowski; Brian L Mark; Robert V Stick; David J Vocadlo |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2007-12-08 |
Journal Detail:
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Title: Journal of the American Chemical Society Volume: 130 ISSN: 1520-5126 ISO Abbreviation: J. Am. Chem. Soc. Publication Date: 2008 Jan |
Date Detail:
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Created Date: 2008-01-02 Completed Date: 2008-01-25 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 7503056 Medline TA: J Am Chem Soc Country: United States |
Other Details:
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Languages: eng Pagination: 327-35 Citation Subset: IM |
Affiliation:
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Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, B.C. Canada. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Binding Sites Humans Methods Molecular Probes / chemical synthesis* Proteomics / methods* Pseudomonas aeruginosa / enzymology* beta-Lactams beta-N-Acetylhexosaminidases / isolation & purification* |
| Chemical | |
Reg. No./Substance:
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0/Molecular Probes; 0/beta-Lactams; EC 3.2.1.52/beta-N-Acetylhexosaminidases |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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