title: Specificity fingerprinting of retaining beta-1,4-glycanases in the Cellulomonas fimi secretome using two fluorescent mechanism-based probes.
abstract: Functional proteomics methods are crucial for activity- and mechanism-based investigation of enzymes in biological systems at a post-translational stage. Glycosidases have central roles in cellular metabolism and its regulation, and their dysfunction can have detrimental effects. These enzymes also play key roles in biomass conversion. A functional profiling methodology was developed for direct, fluorescence-based, in-gel analysis of retaining beta-glycosidases. Two spectrally nonoverlapping fluorescent, mechanism-based probes containing different recognition elements for retaining cellulases and xylanases were prepared. The specificity-based covalent labelling of retaining glycanases by the two probes was demonstrated in model enzyme mixtures. Using the two probes and mass spectrometry, the secretomes of the biomass-converting bacterium Cellulomonas fimi, under induction by different polyglycan growth substrates, were analysed to obtain a specificity profile of the C. fimi retaining beta-glycanases. This is a facile strategy for the analysis of glycosidases produced by biomass-degrading organisms.
authors.affiliation: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, B.C., V6T 1Z1, Canada.
authors.forename: Omid
authors.initials: O
authors.lastname: Hekmat
authors.forename: Christine
authors.initials: C
authors.lastname: Florizone
authors.forename: Young-Wan
authors.initials: YW
authors.lastname: Kim
authors.forename: Lindsay D
authors.initials: LD
authors.lastname: Eltis
authors.forename: R Antony J
authors.initials: RA
authors.lastname: Warren
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D040143:Cellulomonas / Q000187:drug effects / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007425:Intracellular Membranes / Q000187:drug effects / Q000201:enzymology; D015394:Molecular Structure; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D006026:Glycoside Hydrolases; C020668:glycanase