title: Suite of activity-based probes for cellulose-degrading enzymes.
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.
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, 902 Battelle Blvd, Richland, Washington 99352, United States.
authors.forename: Lacie M
authors.initials: LM
authors.lastname: Chauvigné-Hines
authors.forename: Lindsey N
authors.initials: LN
authors.lastname: Anderson
authors.forename: Holly M
authors.initials: HM
authors.lastname: Weaver
authors.forename: Joseph N
authors.initials: JN
authors.lastname: Brown
authors.forename: Phillip K
authors.initials: PK
authors.lastname: Koech
authors.forename: Carrie D
authors.initials: CD
authors.lastname: Nicora
authors.forename: Beth A
authors.initials: BA
authors.lastname: Hofstad
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Wilkins
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Callister
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Wright
mesh_terms: D002482:Cellulose / Q000378:metabolism*; D002853:Chromatography, Liquid; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006868:Hydrolysis; D013058:Mass Spectrometry; D015335:Molecular Probes
chemical_list: D015335:Molecular Probes; D002482:Cellulose; D006026:Glycoside Hydrolases