A mechanism based approach to screening metagenomic libraries for discovery of unconventional glycosidases.

A mechanism based approach to screening metagenomic libraries for discovery of unconventional glycosidases. Angew Chem Int Ed Engl. 2018 Jul 12;: Authors: Withers S, Nasseri S, Betschart L, Opaleva D, Rahfeld P Abstract Functional metagenomics has opened up exciting new opportunities for enzyme discovery. To take advantage of the full potential of this new tool, the design of suitable, selective screens is essential, especially when searching for rare enzymes. In order to identify novel glycosidases that employ cleavage strategies other than the conventional Koshland mechanisms, we needed a suitable screen. Focusing initially on the unsaturated glucuronidases (UGLs) we showed that use of simple aryl glycoside substrates did not allow sufficient discrimination against β-glucuronidases, which are widespread in bacteria. While conventional glycosidases are generally not able to 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 successfully assessed as selective substrates. The generality of the approach was validated by expanding the concept to another family of unconventional glycosidases, the GH4 enzymes. Finally, the utility of these substrates was put to the test and confirmed by screening a small metagenomic library. PMID: 30001477 [PubMed - as supplied by publisher]
Source: Angewandte Chemie - Category: Chemistry Authors: Tags: Angew Chem Int Ed Engl Source Type: research