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 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.

Role of Cytochrome P450 Hydroxylase in the Decreased Accumulation of Vitamin E in Muscle from Turkeys Compared to that from Chickens

“…19 Proteins were digested on-resin with trypsin, and the resulting peptides were collected and analyzed on a LTQ instrument by LC-MS as described previously. 20 Generated MS/MS spectra were searched using the MSGF+ algorithm 21 against the publicly available G. gallus or M. gallopavo translated genome sequences, and rescored using the MS-GF approach. 21 Identified peptides of at least six amino acids in length having MS-GF scores ≤1 × 10 −10 , corresponding to an estimated false discovery rate (FDR) <1% at the peptide level, were used for analysis.…”

Activity-based protein profiling of secreted cellulolytic enzyme activity dynamics in Trichoderma reesei QM6a, NG14, and RUT-C30

“…Following probe incubation, biotin was appended by CuAAC, followed by removal of excess CuAAC reagents by concentration and buffer washing using an Amicon Millipore centrifugal device. 27 Samples were normalized to 1 mg protein before enriching on streptavidin agarose resin. Enriched proteins were washed, digested with trypsin, and vialed for subsequent MS analysis as described previously.…”

“…Enriched proteins were washed, digested with trypsin, and vialed for subsequent MS analysis as described previously. 27 …”

“…26 We recently developed a suite of activity-based probes (ABPs) for glycoside hydrolases, and used them to characterize anaerobic lignocellulose degradation in the bacterium Clostridium thermocellum. 27 Our ABPs are developed as affinity-based inhibitors of glycoside hydrolases (GHs), forming a covalent bond upon reaction with the catalytic domains of target enzymes. Probes are also compatible with copper-mediated azide–alkyne cyclo-additions that permit both fluorescent and LC-MS characterization of ABP-labeled proteins.…”

Progress and prospects for small-molecule probes of bacterial imaging

“…Activitybased probes have been designed to detect a number of bacterial proteins, such as sulfatases 85,86 , serine proteases 87 , the cell wall biosynthetic protein MurA 88 , glycoside hydrolases 89 , redox-sensitive proteins 90,91 and histidine kinases 92 ; however, most of these probes have been used only in gel-based analyses. One additional strategy that has been applied to bacterial imaging is the use of a probe that becomes fluorescent upon turnover by nitroreductase.…”