Activity-based probes for dynamic characterisation of polysaccharide-degrading enzymes

Abstract

Carbohydrate-active enzymes play essential roles in polysaccharide degradation, yet their biochemical characterisation remains challenging - especially in the face of rapidly expanding genomic and structural data. Standard annotations often overlook critical properties such as expression patterns, enzyme stability and substrate specificity, which are key to understanding function in biological and industrial contexts. Activity-based probes (ABPs) offer a direct solution by enabling selective detection of active enzymes within complex systems. This review focuses on ABPs for retaining glycosidases, tracing their development from early applications in medical diagnostics to emerging uses in biomass degradation. We examine recent advances in scaffold design - including fluorosugars, epoxides, aziridines and cyclic sulphates - and their utility in enzyme profiling, inhibitor discovery and biotechnology. Current ABPs remain limited: they cannot yet target inverting enzymes and other classes lacking nucleophilic residues, a gap that may be bridged through computational modelling and AI-guided probe development. Looking forward, integration of ABPs with enzyme engineering and design holds promise for unlocking new classes of biocatalysts tailored for industrial and biomedical use.

Keywords: activity-based probes; biomass conversion; carbohydrate-active enzymes; enzyme inhibitors; glycobiology.

Figure 1:. Sequence similarity network (SSN) reveals subfamily structure within GH30 glycoside hydrolases.

A sequence similarity network (SSN) of 8,066 GH30 proteins was generated using the PFAM identifiers PF02055 and PF14587 via the EFI-EST tool (https://efi.igb.illinois.edu/efi-est/) and visualised in Cytoscape. Nodes represent sequences with ≥80% identity and are colour-coded by CAZy subfamily classification. Characterised sequences appear in dark blue; uncharacterised clusters – highlighting a need for the ABP approach – are grey. GH30_3 (light blue) distinctly separates Bacteria/Archaea and Eukaryota. Subfamilies span a range of activities including but not limited to β−glucosidase, xylosidase, fucosidase, glucuronoxylanase, galactosidase and glucuronidase (see https://www.cazy.org/GH30_activity.html). Edges denote pairwise similarities with e-values <10⁻⁵. The SSN was visualised using Cytoscape (https://cytoscape.org/).

Figure 2:. Activity-based probes (ABPs) and their historical context.

(A) General structure of an ABP: active-site targeting group, recognition element and reporter tag. In two-step labelling the tag could be azide or alkyne for subsequent ‘click’ chemistry. (B) Cravatt’s fluorophosphonate probes, originally developed for serine hydrolases.

Figure 3:. Glycosidase mechanism and design evolution of activity-based probes (ABPs) for covalent glycosidase labelling.

(A) Generic glycosidase mechanism with inversion of anomeric configuration. (B) Generic glycosidase mechanism with net retention of anomeric configuration. Central to this mechanism is nucleophilic attack which is exploited by the ABP approaches described. Note this review will not cover NAD⁺ or neighbouring-group catalytic mechanisms. (C) Vocadlo and Bertozzi’s adaptation of Withers’ 2-fluoro sugars for glycosidase imaging. (D) Cyclophellitol-based ABP schematic for covalent labelling of retaining β-glucosidases.

Figure 4:. Monosaccharide ABPs featuring aziridines and cyclic sulphates for covalent glycosidase labelling.

Representative aziridine-configured monosaccharide probes developed by Overkleeft and co-workers [20,21]. The boxed structure highlights an α-glucoside-configured cyclic sulphate ABP [23], used in antiviral applications targeting ER α-glucosidase II [22].

Figure 5:. Alternative non-cyclophellitol ABPs used to target glycosidases.

Examples of activity-based probes outside the cyclophellitol scaffold class, including quinone methide-releasing reagents and N-haloacetyl sugars, which have been applied to label glycosidases through diverse covalent mechanisms. ABPs, activity-based probes.

Figure 6:. Activity-based probes (ABPs) enable structural and temporal mapping of xyloglucan degradation.

(A) Representative xyloglucan structure: a β-1,4-glucan backbone with diverse side chains, including α-1,6-linked xylose and extended decorations (not all substitutions co-occur in nature, the scheme is for illustrative purposes only; see [54] for discussion of xyloglucan structure). (B) ABP panel used to target multiple retaining glycosidases involved in xyloglucan breakdown. (C) Multiplex ABP profiling of Cellvibrio japonicus enzyme expression over time, under different carbon sources (xyloglucan oligosaccharides, polymeric xyloglucan or glucose). Adapted from ref. [55].

Figure 7:. Probing α-amylase specificity and discovery using a modular activity-based probe (ABP) toolbox.

(A) Structures of starch and α-amylase-targeted ABPs. (B) Labelling of α-amylases in complex biological samples with probe 3. Adapted from [57]. (C) Substrate preferences of human gut α-amylases revealed by probes 3, 10 and 14. (D) Active site covalent binding of inhibitor 9 to Ruminococcus bromii amylase RbAmy5. (E) Discovery of novel α-amylases from compost using pull-down assays; volcano plots highlight proteins significantly enriched by probes 4, 12 and 16. C–E adapted from [58].