Search Results (2)

Glucuronides hydrolysis by intestinal microbial β-Glucuronidases (GUS) is an important procedure for many endogenous and exogenous compounds. The purpose of this study is to determine the impact of experimental conditions on glucuronide hydrolysis by intestinal microbial GUS. Standard probe 4-Nitrophenyl β-D-glucopyranoside (pNPG) and a natural glucuronide wogonoside were used as the model compounds. Feces collection time, buffer conditions, interindividual, and species variations were evaluated by incubating the substrates with enzymes. The relative reaction activity of pNPG, reaction rates, and reaction kinetics for wogonoside were calculated. Fresh feces showed the highest hydrolysis activities. Sonication increased total protein yield during enzyme preparation. The pH of the reaction system increased the activity in 0.69–1.32-fold, 2.9–12.9-fold, and 0.28–1.56-fold for mouse, rat, and human at three different concentrations of wogonoside, respectively. The Vmax for wogonoside hydrolysis was 2.37 ± 0.06, 4.48 ± 0.11, and 5.17 ± 0.16 μmol/min/mg and Km was 6.51 ± 0.71, 3.04 ± 0.34, and 0.34 ± 0.047 μM for mouse, rat, and human, respectively. The inter-individual difference was significant (4–6-fold) using inbred rats as the model animal. Fresh feces should be used to avoid activity loss and sonication should be utilized in enzyme preparation to increase hydrolysis activity. The buffer pH should be appropriate according to the species. Inter-individual and species variations were significant. Full article

Non-typhoidal Salmonella ingeniously scavenges energy for growth from tyramine (TYR) and d-glucuronic acid (DGA), both of which occur in the host as the metabolic byproducts of the gut microbial metabolism. A critical first step in energy scavenging from TYR and DGA in Salmonella involves TYR-oxidation via TYR-oxidoreductase and production of free-DGA via β-glucuronidase (GUS)-mediated hydrolysis of d-glucuronides (conjugated form of DGA), respectively. Here, we report that Salmonella utilizes TYR and DGA as sole sources of energy in a serotype-independent manner. Using colorimetric and radiometric approaches, we report that genes SEN2971, SEN3065, and SEN2426 encode TYR-oxidoreductases. Some Salmonella serotypes produce GUS, thus can also scavenge energy from d-glucuronides. We repurposed phenelzine (monoaminoxidase-inhibitor) and amoxapine (GUS-inhibitor) to inhibit the TYR-oxidoreductases and GUS encoded by Salmonella, respectively. We show that phenelzine significantly inhibits the growth of Salmonella by inhibiting TYR-oxidoreductases SEN2971, SEN3065, and SEN2426. Similarly, amoxapine significantly inhibits the growth of Salmonella by inhibiting GUS-mediated hydrolysis of d-glucuronides. Because TYR and DGA serve as potential energy sources for Salmonella growth in vivo, the data and the novel approaches used here provides a better understanding of the role of TYR and DGA in Salmonella pathogenesis and nutritional virulence. Full article