Fig. 2
Structural analysis of the catalytic site in Osm1. a An omit density map contoured at the 1−σ level around the active site. The flavin and clamp domains are shown in blue and pink, respectively. The active site is shown by the red dotted rectangle. The FAD and substrate-binding site is magnified for better visualization. b A 2D FAD-Osm1 interaction diagram. The residues involved in the FAD interaction are indicated. Green-colored residues are involved in hydrogen bonding. The green dotted lines indicate hydrogen bonds. The 2D interaction diagram was generated by Ligplot30. c The substrate-binding site, magnified to show a clear electron density map. d The 2D substrate (fumarate)-Osm1 interaction diagram. The residues involved in the substrate accommodation are indicated. e Structural comparison of the active site of Osm1 with Fcc3 fumarate reductase from Shewanella frigidimarina (1QJD). The location of six completely conserved amino acids which may be critical for Osm1 activity, H281 (H365), E301 (E378), R304 (R381), R326 (R402), H435 (H504), and R477 (R544), are indicated. f Electrons can be transferred from FADH2 to fumarate in the absence of oxygen. The fumarate reductase activity of Osm1 can be assessed by monitoring the oxidation of free FADH2 based on the absorbance of FAD at 450 nm. g Comparison of fumarate reductase activities of Osm1 wild type and its catalytic mutants. Oxidation of FADH2 was monitored under anaerobic environment induced by sodium dithionite. A representative reaction curve among three replications was provided. h Bar graph of fumarate reductase activity of Osm1 wild type and its mutants. The corresponding dot plots were overlaid in the bar charts. The reaction rates were calculated based on reaction curves (n = 3, error bar was derived bystandard error)
