Activity-Based Metaproteomics Driven Discovery and Enzymological Characterization of Potential α-Galactosidases in the Gut Microbiome

Abstract

The gut microbiota offers an extensive resource of enzymes, but many remain uncharacterized. To distinguish activities of similar annotated proteins and mine the potentially applicable ones in the microbiome, we developed an effective Activity-Based Metaproteomics (ABMP) strategy using a specific activity-based probe (ABP) to screen the entire gut microbiome for directly discovering active enzymes. Using an activity-based cyclophellitol aziridine probe specific to α-galactosidases (AGAL), we successfully identified and characterized several gut microbiota enzymes possessing AGAL activities. Cryo-electron microscopy analysis of a newly characterized enzyme (AGLA5) revealed the covalent binding conformations between the AGAL5 active site and the cyclophellitol aziridine ABP, which could provide insights into the enzyme’s catalytic mechanism. The four newly characterized AGALs have diverse potential activities, including raffinose family oligosaccharides (RFOs) hydrolysis and enzymatic blood group transformation. Collectively, we present a ABMP platform for facilitating gut microbiota enzyme discovery, biochemical activity annotations and potential industrial or biopharmaceutical applications.