A Mechanism‐Based Approach to Screening Metagenomic Libraries for Discovery of Unconventional Glycosidases

Abstract

Functional metagenomics has opened new opportunities for enzyme discovery. To exploit the full potential of this new tool, the design of selective screens is essential, especially when searching for rare enzymes. To identify novel glycosidases that employ cleavage strategies other than the conventional Koshland mechanisms, a suitable screen was needed. Focusing on the unsaturated glucuronidases (UGLs), it was found that use of simple aryl glycoside substrates did not allow sufficient discrimination against β-glucuronidases, which are widespread in bacteria. While conventional glycosidases cannot generally hydrolyze thioglycosides efficiently, UGLs follow a distinct mechanism that allows them to do so. Thus, fluorogenic thioglycoside substrates featuring thiol-based self-immolative linkers were synthesized and assessed as selective substrates. The generality of the approach was validated with another family of unconventional glycosidases, the GH4 enzymes. Finally, the utility of these substrates was tested by screening a small metagenomic library.

Biocatalysis

“…2c). Functionalbased screening can incorporate the use of colorimetric assays [30][31][32] , mechanism-based probes 33 and/or droplet microfluidics 34 . Researchers are looking for enzymes in these libraries that demonstrate catalysis of the desired…”

Discovery of Novel Biosynthetic Gene Cluster Diversity From a Soil Metagenomic Library

“…Nevertheless, the vast majority of environmental microbiota can only be exploited for their natural products via culture-independent approaches (Milshteyn et al, 2014). Indeed, functional screening of metagenomic clone libraries has led to the discovery of structurally and functionally diverse antimicrobial compounds Brady, 2013, 2014a;Hover et al, 2018;Nasrin et al, 2018), bioactive compounds (Fisch et al, 2009;Zimmermann et al, 2009;Chang and Brady, 2013;Owen et al, 2015), novel enzymes (Wang et al, 2010;Cheng et al, 2017;Kusnezowa and Leichert, 2017;Lewin et al, 2017;Nasseri et al, 2018), and novel promoters (Westmann et al, 2018).…”

High‐Throughput Approaches in Carbohydrate‐Active Enzymology: Glycosidase and Glycosyl Transferase Inhibitors, Evolution, and Discovery

“…[61]), F) by generation of aproduct not recognised by transporters (Ref. [83]). [68]), and (H) by requiring initial cleavage of athioglycosidic linkage (Ref.…”

“…Such an approach based on fluorogenic substrates is typically able to find enzymes with the desired activity but for common reactions often will find the same or similar enzymes each time.I no rder to guide as creen to find more esoteric enzymes,e specially those that operate through ad ifferent mechanism, [82] aset of substrates were developed that contain Angewandte Chemie an additional linker between the sugar and the fluorophore (Figure 3H). [83] Important in this design is that fluorescence is generated only on cleavage of athioglycosidic bond, which is typically intractable to glycoside hydrolases that operate through the more common retaining or inverting mechanisms. Application of these substrates to as mall test metagenomic library derived from beaver fecal matter showed clear discrimination between desired and undesired activities,while simple substrates without the thioglycosidic immolative linker showed false positives from side activities of more common enzymes.Itisworth noting, however,that while this substrate is cleverly designed to find certain types of new reactivities,it has its own limitations in which types of new mechanisms it could find.…”