Orthogonal Active-Site Labels for Mixed-Linkage endo-β-Glucanases

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.