FIGURE 4: DMB is a competitive inhibitor of the NAD(P)H:flavin reductase (Fre) enzyme. (A) Initial reaction velocities were calculated using purified S. Typhimurium Fre enzyme as described in Materials and Methods. Reaction rates at a fixed concentration of FMN (1 mM) and different concentrations of DMB or other bases (as indicated) are shown. (B) Double-reciprocal plot of Fre initial rates at varying concentrations of FMN and different fixed concentrations of DMB as indicated. Error bars represent standard deviation of three technical replicates and each experiment was replicated twice, with a representative graph shown. K_i was calculated by a competitive inhibition model fit using Prism (GraphPad, v9). Standard deviation of two independent experiments is shown. An unpaired Student's t test was performed to determine statistical significance; ns, not significant. Statistical compassions were performed against the no-treatment control unless otherwise shown in the Figure.