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.

Functional metagenomics reveals an alternative, broad-specificity pathway for metabolism of carbohydrates in human gut commensal bacteria

“…We synthesized the seven fluorogenic thioglycoside substrates shown in Fig. 2, based on β-glucose, β-galactose, α-mannose, β-glucuronic acid, 4,5-unsaturated β-glucuronic acid, β-N-acetylglucosamine and β-6-phosphoglucose, based on previously reported methods 7,11 and as described in the SI (Schemes S1-4). These substrates cover a broad spectrum of commonly occurring glycosides as well as two ( 5 and 7 ) as controls since enzymes from GH4 (6-phospho-β-glucosidase) and/or GH88/105 (α-4-5-ene-glucuronidase) were likely to be present in our libraries.…”

“…As screening substrates for the discovery of new mechanistic classes of glycosidases, we used the fluorogenic alkylthioglycosides (Fig. 1b) that we had previously 7 shown to not be hydrolysed by Koshland glycosidases, yet cleaved efficiently by non-Koshland glycosidases from CAZy 8 families GH4/109 9 and GH88/105 10 . These substrates will therefore detect not only thioglycosidases but also other possible glycoside-cleaving enzymes that do not use Koshland mechanisms.…”

Molecular biology interventions for activity improvement and production of industrial enzymes

“…Another approach makes use of substrates with self immolative linkers, specifically for screening of unsaturated glucuronidases (Nasseri et al, 2018). Substrate induced gene expression screening (SIGEX), which contains GFP as a reporter gene is another interesting approach to identify novel enzymes from metagenomic libraries.…”

Challenges of Biomass Utilization for Bioenergy in a Climate Change Scenario

“…As previously discussed, glucuronic acid residuals are also part of hemicelluloses, as glucuronans and glucuronoglycan. β-glucuronidases (EC 3.2.1.31) hydrolyze glucuronic acid [ 96 ].…”