Suite of Activity-Based Probes for Cellulose-Degrading Enzymes

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• 作者：Lacie M. Chauvign茅-Hines ; Lindsey N. Anderson ; Holly M. Weaver ; Joseph N. Brown ; Phillip K. Koech ; Carrie D. Nicora ; Beth A. Hofstad ; Richard D. Smith ; Michael J. Wilkins ; Stephen J. Callister ; Aaron T. Wright
• 刊名：The Journal of the American Chemical Society
• 出版年：2012
• 出版时间：December 19, 2012
• 年：2012
• 卷：134
• 期：50
• 页码：20521-20532
• 全文大小：613K
• 年卷期：v.134,no.50(December 19, 2012)
• ISSN：1520-5126

文摘

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鈥搈ass spectrometry (LC鈥揗S) 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.