Fig. 6: Identification of FAD-binding region and electron shuttling path through MD simulation and mutant characterization of sm1.
a In silico superposition of FAD domain in native MMOR (pink) and RFAD-ΔHα in sm1 (cyan). In the native MMOR, FAD and FAD-binding residues are marked by red sticks, while in RFAD-ΔHα, they are marked by blue sticks, showing that FAD is localized much closer to the diiron center in RFAD-ΔHα. b, c Closer view of FAD in the hydrophobic pocket of RFAD-ΔHα, shown by in silico imaging. The putative residues comprising the hydrophobic pocket is visualized by magenta sticks in (b) and by a reddish groove on the molecular surface in (c), where hydrophobicity is indicated by a color gradient that ranges from red (representing hydrophobic) to blue (representing hydrophilic). In (b), the residues potentially involved in the electron shuttling are shown as blue sticks, and the diiron ligand residues are shown as pink sticks. All results were obtained from isobaric-isothermal MD simulations of RFAD-ΔHα equilibrated in water medium at 300 K and 1 bar for 500 ns. d Primary structure of RFAD-ΔHα. The hydrophobic residues involved in FAD-binding residues, diiron ligands, and those potentially involved in electron shuttling are color-coded in red, pink, and blue, respectively. e Table showing the sm1 mutants in the putative FAD-binding region (sm1-FADmut) and electron transport path (sm1-emut1 to sm1-emut4). Sky-blue symbols indicate hydrophilic amino acids, and red symbols indicate hydrophobic amino acids. f Fluorescence intensity from FAD in sm1 and the mutants of (e) (sm1-FADmut and sm1-emut1 to sm1-emut4). N = 3 independent experiments. Mean ± s.d. g Cumulative amount of methanol produced by sm1 and the mutants of (e). N = 3 independent experiments. Mean ± s.d. Source Data are provided with this paper.
