Urolithin 9-dehydroxylase from Enterocloster bolteae JCM 12243T catalyzing regiospecific dehydroxylation of urolithins

Abstract

Urolithins are metabolites derived from ellagic acid, a compound that is present in many plants, including pomegranate, via the action of gut microorganisms. Among them, urolithin A exhibits antioxidant and anti-inflammatory activity and promotes autophagy, making it a potential contributor to human health. Enterocloster bolteae JCM 12243^T, isolated from human feces, is able to convert urolithin C into urolithin A. In previous studies, the urolithin C dehydroxylase encoded by the ucdCFO (or ucdhABC) from E. bolteae DSM 15670^T (=JCM 12243^T) was identified through proteomic or transcriptomic analyses. In contrast, we aimed to identify the enzymes responsible for the reaction using through purification from cell extracts of the wild type active strain. Our results show that UcdCFO/UcdhABC is the sole catalyst of urolithin C dehydroxylation to urolithin A in this bacterium. To further characterize the enzyme, we obtained it from the Rhodococcus erythropolis L88 transformant expressing the codon-optimized genes and analyzed its properties. The purified enzyme catalyzed dehydroxylation at the 9-position of five urolithins, indicating that the enzyme is a urolithin 9-dehydroxylase. Kinetic analysis indicated that the enzyme has a high catalytic activity toward urolithin M6 with almost the same activities to urolithin M5 and urolithin C. This enzyme exhibited maximum activity for the conversion of urolithin C into urolithin A between pH 6.5 and 7.5 and at 37 °C. This findings provide valuable information for the efficient fermentation-based production of urolithin A, which exerts health-promoting effects.