Structural Basis of Microbial β-Glucosidase Processing and Reactivation of Glycosylated Phytochemicals in the Human Gut

A large number of health promoting plant chemicals obtained by humans through their diet arrive as glucoside conjugates. These conjugates are primarily processed by gut microbial enzymes, suggesting the importance of understanding the reactions performed by those proteins. Glucosidases are enzymes produced by gut microbes that function to cleave glucose from glycosylated phytochemicals encountered in the human gut, consequently reactivating these metabolites and enhancing their health benefits, including antibacterial, anti-inflammatory, and antioxidant effects. Therefore, this work explored the unique structures and substrate specificities of β-glucosidases produced by different gut microbes to uncover which bacterial species produce β-glucosidases most essential for promoting the benefits of specific metabolites. To investigate diverse glucosidase functions, we determined the specific activity of GH3 β-glucosidases from six different bacterial species with a panel of seven relevant glycosylated phytochemical substrates using plate reader enzyme kinetic assays. Additionally, X-ray crystallography was employed to solve the protein structure of the P. dorei glucosidase. AlphaFold models of unsolved β-glucosidases were used to make structural comparisons for rationalization of their unique enzymatic activities. Overall, this work aims to uncover the role of glucosidases in supporting human health through phytochemical reactivation.