Orthogonal Active-Site Labels for Mixed-Linkage endo-β-Glucanases.
Namrata JainKazune TamuraGuillaume DéjeanFilip Van PetegemHarry BrumerPublished in: ACS chemical biology (2021)
Small molecule irreversible inhibitors are valuable tools for determining catalytically important active-site residues and revealing key details of the specificity, structure, and function of glycoside hydrolases (GHs). β-glucans that contain backbone β(1,3) linkages are widespread in nature, e.g., mixed-linkage β(1,3)/β(1,4)-glucans in the cell walls of higher plants and β(1,3)glucans in yeasts and algae. Commensurate with this ubiquity, a large diversity of mixed-linkage endoglucanases (MLGases, EC 3.2.1.73) and endo-β(1,3)-glucanases (laminarinases, EC 3.2.1.39 and EC 3.2.1.6) have evolved to specifically hydrolyze these polysaccharides, respectively, in environmental niches including the human gut. To facilitate biochemical and structural analysis of these GHs, with a focus on MLGases, we present here the facile chemo-enzymatic synthesis of a library of active-site-directed enzyme inhibitors based on mixed-linkage oligosaccharide scaffolds and N-bromoacetylglycosylamine or 2-fluoro-2-deoxyglycoside warheads. The effectiveness and irreversibility of these inhibitors were tested with exemplar MLGases and an endo-β(1,3)-glucanase. Notably, determination of inhibitor-bound crystal structures of a human-gut microbial MLGase from Glycoside Hydrolase Family 16 revealed the orthogonal labeling of the nucleophile and catalytic acid/base residues with homologous 2-fluoro-2-deoxyglycoside and N-bromoacetylglycosylamine inhibitors, respectively. We anticipate that the selectivity of these inhibitors will continue to enable the structural and mechanistic analyses of β-glucanases from diverse sources and protein families.
Keyphrases
- small molecule
- endothelial cells
- genome wide
- single cell
- systematic review
- randomized controlled trial
- stem cells
- squamous cell carcinoma
- induced pluripotent stem cells
- protein protein
- mesenchymal stem cells
- risk assessment
- hydrogen peroxide
- computed tomography
- pluripotent stem cells
- hepatitis c virus
- drinking water
- dna methylation
- bone marrow
- high resolution
- cancer therapy
- molecularly imprinted
- saccharomyces cerevisiae
- gold nanoparticles
- drug delivery
- tissue engineering
- combination therapy
- life cycle