A Mechanism-Based Approach to Screening Metagenomic Libraries for Discovery of Unconventional Glycosidases.
Angew Chem Int Ed Engl
; 57(35): 11359-11364, 2018 08 27.
Article
em En
| MEDLINE
| ID: mdl-30001477
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 ß-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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Metagenômica
/
Glicosídeo Hidrolases
Tipo de estudo:
Diagnostic_studies
/
Screening_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Angew Chem Int Ed Engl
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Canadá