Nitroreductase from Enterococcus faecalis catalyzes the metabolic activation of sennoside A in the colon via a unique CC reductive cleavage

Human gut bacteria are involved in the metabolic activation of sennoside A (SA), a widely used laxative. However, the molecular mechanism by which SA is converted to its active form, rheinanthrone, remains unclear. In this study, we propose a novel biochemical mechanism in which bacterial nitroreductases catalyze the conversion of SA to rheinanthrone via a unique CC reductive cleavage. Through functional screening of a type-strain library, seven representative anaerobic bacterial strains were examined, among which Enterococcus faecalis exhibited significant reductive cleavage activity. Both in vitro and in vivo experiments confirmed that this SA-reducing phenotype in E. faecalis could be abolished by dicoumarol, a known nitroreductase inhibitor. Using a previously characterized model enzyme, BpNfrA, as a probe, we identified a homologous nitroreductase in E. faecalis, designated EfNfrA, and confirmed its reductive cleavage activity through in vitro assays. Mechanistic insights into this FMN-dependent reductase were further elucidated through molecular docking and molecular dynamics simulations. To our knowledge, this is the first report demonstrating that a specific bacterial nitroreductase is responsible for the metabolic activation of SA in the gut. These findings enhance our understanding of the pharmacology of SA and provide a model for drug metabolism mediated by the gut microbiota.