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

ACS Central Science, vol. 9, pp. 2306–2314

Abstract

High Resolution Image Download MS PowerPoint Slide 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.

Authors 9

1. Nicholas G. S. McGregor

   University of York

   Affiliation as printed

   Department of Chemistry, The University of York, Heslington, York YO10 5DD, United Kingdom

2. Casper de Boer Aachen

   Leiden University

   Affiliation as printed

   Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA, Leiden, The Netherlands

3. Quentin P. O. Foucart

   University of York

   Affiliation as printed

   Department of Chemistry, The University of York, Heslington, York YO10 5DD, United Kingdom

4. Thomas J. M. Beenakker Aachen

   Leiden University

   Affiliation as printed

   Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA, Leiden, The Netherlands

5. Wendy A. Offen

   University of York

   Affiliation as printed

   Department of Chemistry, The University of York, Heslington, York YO10 5DD, United Kingdom

6. Jeroen D. C. Codée Aachen

   Leiden University

   Affiliation as printed

   Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA, Leiden, The Netherlands

7. Lianne I. Willems

   University of York

   Affiliation as printed

   York Structural Biology Laboratory and York Biomedical Research Institute, Department of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom

8. Herman S. Overkleeft corresponding Aachen

   Leiden University

   Affiliation as printed

   Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA, Leiden, The Netherlands

9. G.J. Davies corresponding

   University of York

   Affiliation as printed

   Department of Chemistry, The University of York, Heslington, York YO10 5DD, United Kingdom