Synthesis and use of mechanism-based protein-profiling probes for retaining beta-D-glucosaminidases facilitate identification of Pseudomonas aeruginosa NagZ.
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.
Stubbs
Keith A
KA
Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, B.C. Canada.
Scaffidi
Adrian
A
Debowski
Aleksandra W
AW
Mark
Brian L
BL
Stick
Robert V
RV
Vocadlo
David J
DJ
D001665:Binding Sites; D006801:Humans; D008722:Methods; D015335:Molecular Probes / Q000138:chemical synthesis*; D040901:Proteomics / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D047090:beta-Lactams; D001619:beta-N-Acetylhexosaminidases / Q000302:isolation & purification*
D015335:Molecular Probes; D047090:beta-Lactams; D001619:beta-N-Acetylhexosaminidases