Fig. 4: Mechanism of napabucasin bioactivation by OOR.
a Cryo-EM structure of the apo-form OOR. The images present both the front and top views of the cryo-EM map, as well as the cartoon representation of OOR in its apo state. Each protomer consists of four subunits: OorA (dark blue), OorB (dark green), OorC (purple), and OorD (chocolate). Protomer B is illustrated in lighter colors to facilitate differentiation from protomer A. b Electron transfer pathway within protomer A. TPP is shown in sticks while proximal SF4, medial SF4, and distal SF4 are shown as spheres. The inter-ligand distances are indicated with dotted lines, and the respective measurements are annotated. c Cryo-EM structure of OOR in complex with NPB. d Interactions of NPB with the nearby residues. The threshold of the local mesh map of NPB is set to 0.25. Distances between NPB and proximal SF4, medial SF4 are marked. e MST measurements of the OOR mutant proteins binding to NPB at various concentrations (0.488 nM-8 μM). Normalized fluorescence (ΔFnorm) was plotted against the concentration of NPB or metronidazole (MTZ). The Kd values are shown in each plot. f Relative NPB or menaquinone 6 (MK-6) reductase activity of the OOR mutant proteins. Data are represented as mean ± SEM. g Resistant spot dilution assays for the parent strain BHKHpS42 (OOR), BHKHpS43 (L44A), BHKHpS44 (K46A), and BHKHpS45 (L44A K46A) on Columbia blood agar plates containing indicated concentrations of NPB. MTZ addition was used as a control. Data are represented as mean ± SEM (n = 3 biological replicates) in (e, f, h). h Detection of ROS levels in the parent strain BHKHpS42 (OOR), BHKHpPS43 (L44A), BHKHpS44 (K46A), and BHKHpS45 (L44A K46A) after treatment with NPB for 4 h. Statistical significance was calculated using a one-way ANOVA with Tukey’s post-hoc test. Source data are provided as a Source Data file.
