Document Detail


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