A Multiplexing Activity-Based Protein-Profiling Platform for Dissection of a Native Bacterial Xyloglucan-Degrading System

Bacteria and yeasts
grow on biomass polysaccharides by
expressing
and excreting a complex array of glycoside hydrolase (GH) enzymes.
Identification and annotation of such GH pools, which are valuable
commodities for sustainable energy and chemistries, by conventional
means (genomics, proteomics) are complicated, as primary sequence
or secondary structure alignment with known active enzymes is not
always predictive for new ones. Here we report a “low-tech”,
easy-to-use, and sensitive multiplexing activity-based protein-profiling
platform to characterize the xyloglucan-degrading GH system excreted
by the soil saprophyte, Cellvibrio japonicus, when grown on xyloglucan. A suite of activity-based probes bearing
orthogonal fluorophores allows for the visualization of accessory exo-acting glycosidases, which are then identified using
biotin-bearing probes. Substrate specificity of xyloglucanases is
directly revealed by imbuing xyloglucan structural elements into bespoke
activity-based probes. Our ABPP platform provides a highly useful
tool to dissect xyloglucan-degrading systems from various sources
and to rapidly select potentially useful ones. The observed specificity
of the probes moreover bodes well for the study of other biomass polysaccharide-degrading
systems, by modeling probe structures to those of desired substrates.