As the scope of modern genomics technologies increases, so does the need for informative chemical tools to study functional biology. Activity-based probes (ABPs) provide a powerful suite of reagents to probe the biochemistry of living organisms. These probes, featuring a specificity motif, a reactive chemical group and a reporter tag, are opening-up large swathes of protein chemistry to investigation in vitro, as well as in cellular extracts, cells and living organisms in vivo. Glycoside hydrolases, by virtue of their prominent biological and applied roles, provide a broad canvas on which ABPs may illustrate their functions. Here we provide an overview of glycosidase ABP mechanisms, and review recent ABP work in the glycoside hydrolase field, encompassing their use in medical diagnosis, their application for generating chemical genetic disease models, their fine-tuning through conformational and reactivity insight, their use for high-throughput inhibitor discovery, and their deployment for enzyme discovery and dynamic characterization. Highlights  Glycosidases carry out many essential functions across all domains of life  Activity-based probes can interrogate complex biological samples for glycosidase activity  Glycosidase probes can characterize enzymes of biomedical/biotechnological interest  Analysis of conformational itineraries may inform the design of future probes.  Activity-based probes assist high-throughput discovery of inhibitors

Background: DypB, a Dyp-type peroxidase, oxidizes Mn(II) and transforms lignin. Results: DypB forms a stable Compound I that rapidly decays to Compound II in the D153A and N246A but is undetectable in the R244L variant. Conclusion: The requirement of Arg-244 but not Asp-153 to form Compound I indicates that DyPs modulate the peroxidative cycle differently than plant peroxidase. Significance: Understanding DyPs helps harness their biotechnological potential.

Heparan sulfate proteoglycans (HSPGs) mediate essential interactions throughout the extracellular matrix (ECM), providing signals that regulate cellular growth and development. Altered HSPG composition during tumorigenesis strongly aids cancer progression. Heparanase (HPSE) is the principal enzyme responsible for extracellular heparan sulfate catabolism and is markedly up-regulated in aggressive cancers. HPSE overactivity degrades HSPGs within the ECM, facilitating metastatic dissemination and releasing mitogens that drive cellular proliferation. Reducing extracellular HPSE activity reduces cancer growth, but few effective inhibitors are known, and none are clinically approved. Inspired by the natural glycosidase inhibitor cyclophellitol, we developed nanomolar mechanism-based, irreversible HPSE inhibitors that are effective within physiological environments. Application of cyclophellitol-derived HPSE inhibitors reduces cancer aggression in cellulo and significantly ameliorates murine metastasis. Mechanism-based irreversible HPSE inhibition is an unexplored anticancer strategy. We demonstrate the feasibility of such compounds to control pathological HPSE-driven malignancies.

Functional metagenomics has emerged as a powerful method for gene model validation and enzyme discovery from natural and human engineered ecosystems. Here we report development of a high-throughput functional metagenomic screen incorporating bioinformatic and biochemical analyses features. A fosmid library containing 6144 clones sourced from a mining bioremediation system was screened for cellulase activity using 2,4-dinitrophenyl β-cellobioside, a previously proven cellulose model substrate. Fifteen active clones were recovered and fully sequenced revealing 9 unique clones with the ability to hydrolyse 1,4-β-D-glucosidic linkages. Transposon mutagenesis identified genes belonging to glycoside hydrolase (GH) 1, 3, or 5 as necessary for mediating this activity. Reference trees for GH 1, 3, and 5 families were generated from sequences in the CAZy database for automated phylogenetic analysis of fosmid end and active clone sequences revealing known and novel cellulase encoding genes. Active cellulase genes recovered in functional screens were subcloned into inducible high copy plasmids, expressed and purified to determine enzymatic properties including thermostability, pH optima, and substrate specificity. The workflow described here provides a general paradigm for recovery and characterization of microbially derived genes and gene products based on genetic logic and contemporary screening technologies developed for model organismal systems.