Specificity Fingerprinting of Retaining β-1,4-Glycanases in the Cellulomonas fimi Secretome Using Two Fluorescent Mechanism-Based Probes

• Published in: Chembiochem : a European journal of chemical biology Vol. 8; no. 17; pp. 2125 - 2132
• Main Authors: Hekmat, Omid, Florizone, Christine, Kim, Young-Wan, Eltis, Lindsay D, Warren, R. Antony J, Withers, Stephen G
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley-VCH Verlag 23.11.2007; WILEY-VCH Verlag; WILEY‐VCH Verlag
• Subjects: affinity fingerprinting; Cellulomonas - drug effects; Cellulomonas - enzymology; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; enzyme specificity; Fluorescent Dyes - chemistry; fluorescent probes; glycosidases; Glycoside Hydrolases - antagonists & inhibitors; Glycoside Hydrolases - metabolism; Intracellular Membranes - drug effects; Intracellular Membranes - enzymology; Molecular Structure; proteomics; Sensitivity and Specificity; Substrate Specificity
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.200700481

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 β-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 β-glycanases. This is a facile strategy for the analysis of glycosidases produced by biomass-degrading organisms.