Glycosylated cyclophellitol-derived activity-based probes and inhibitors for cellulases

Abstract

New cyclophellitol-derived activity-based probes enable the sensitive detection and identification of cellulases.

Activity-based protein profiling reveals dynamic substrate-specific cellulase secretion by saprotrophic basidiomycetes

“…As an initial test of specificity, we compared activity towards 4MU-GG and 4-methylumbelliferyl xylobioside (4MU-Xyl2), finding no detectable activity towards 4MU-Xyl2 among LsGH5_5A and TlGH12A, and a strong preferential activity towards 4MU-Xyl2 for LsGH10A (Additional file 11 : Table S2). Using 4MU-GG as substrate, we measured inhibition of LsGH5_5A, LsGH10A, and TlGH12A over time by glucosyl-β(1,4)-cyclophellitol [ 36 ] (GGcyc) at inhibitor concentrations as high as 50 μM under optimal buffer conditions (see Additional file 11 : Figs. S13 and S14 for effects of buffer and pH on enzyme activity).…”

“…ABP-Xyn, an established N -alkyl aziridine probe bearing a Cy5 + tag, was used to label endo -β-xylanases [ 35 ]. Endo -β-glucanase probe CB644 was prepared through click modification of ABP-Cel with Cy3 + alkyne in place of previously reported Cy5 + alkyne [ 36 ].…”

“…Briefly, enzyme in 100 mM pH 4.0 NaOAc buffer was mixed 1:1, to a final concentration selected to hydrolyse ~ 5% of the substrate over 2 h, with inhibitor and 0.4 mM substrate (diluted from 100 mM in DMSO) in water. Inhibitor concentrations from 0 to 50 μM or 0 to 25 μM were monitored for fluorescence continuously for up to 2 h. To test enzyme recognition specificity, inhibition was measured with glucosyl-β(1,4)-cyclophellitol (GGcyc) [ 36 ] or xylosyl-β(1,4)-xylocyclophellitol (XXcyc) [ 35 ]. To test the impact of the different linker chemistries, inhibition kinetics were also measured using Biotin-ABP-Xyn [ 35 ] and Biotin-ABP-Cel [ 36 ].…”

“…Inhibitor concentrations from 0 to 50 μM or 0 to 25 μM were monitored for fluorescence continuously for up to 2 h. To test enzyme recognition specificity, inhibition was measured with glucosyl-β(1,4)-cyclophellitol (GGcyc) [ 36 ] or xylosyl-β(1,4)-xylocyclophellitol (XXcyc) [ 35 ]. To test the impact of the different linker chemistries, inhibition kinetics were also measured using Biotin-ABP-Xyn [ 35 ] and Biotin-ABP-Cel [ 36 ].…”

Activity-Based Protein Profiling of Retaining α-Amylases in Complex Biological Samples

“… 28 − 32 More recently, we expanded the methodology toward retaining endoglycosidases in our work on xylobiose-configured ABPs to investigate endo -acting xylanases in Aspergillus secretomes. 33 , 34 Here, we reveal the efficacy of retaining glycosidase ABPP by the design and chemical synthesis of a panel of maltobiose epi -cyclophellitol derived retaining α-amylase inhibitors and ABPs, the kinetic and structural analysis of their interactions with microbial α-amylases, and the application of these probes for rapid detection and identification of α-amylases in fungal secretomes, mouse tissue, and human saliva. Our work complements a recent 24 study by Withers and colleagues, who reported a chemoenzymatic synthesis of maltobiose epi -cyclophellitol, to which we could match our chemically derived material and as well our structural work on α-amylase-inhibitor complexes.…”