U.S. Department of Energy

Pacific Northwest National Laboratory

Suite of activity-based probes for cellulose-degrading enzymes.

TitleSuite of activity-based probes for cellulose-degrading enzymes.
Publication TypeJournal Article
Year of Publication2012
AuthorsChauvignè-Hines LM, Anderson LN, Weaver HM, Brown JN, Koech PK, Nicora CD, Hofstad BA, Smith RD, Wilkins MJ, Callister SJ, Wright AT
JournalJ Am Chem Soc
KeywordsCellulose, Chromatography, Liquid, Glycoside Hydrolases, Hydrolysis, Mass Spectrometry, Molecular Probes
Abstract

Microbial glycoside hydrolases play a dominant role in the biochemical conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits containing cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degradation of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome-producing bacterium that couples rapid reproduction rates to highly efficient degradation of crystalline cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycone, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chemistry and to increase enzyme active site inclusion for liquid chromatography-mass spectrometry (LC-MS) analysis. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes, in addition to a large collection of integral cellulosome-containing proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose-degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic microbial cellulose-degrading systems and facilitates a greater understanding of the organismal role associated with biofuel development.

DOI10.1021/ja309790w
PubMed ID23176123
PubMed Central IDPMC3538167
Grant List5P41RR018522 / RR / NCRR NIH HHS / United States
8P41GM103493-10 / GM / NIGMS NIH HHS / United States
P41 GM103493 / GM / NIGMS NIH HHS / United States
P41 RR018522 / RR / NCRR NIH HHS / United States
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