Stereospecificity of hydride transfer and molecular docking in FMN‐dependent NADH‐indigo reductase of Bacillus smithii

In this study, we investigated the stereospecificity of hydride transfer from NADH to flavin mononucleotide (FMN) in reactions catalyzed by the FMN‐dependent NADH‐indigo reductase expressed by thermophilic Bacillus smithii. We performed 1H‐NMR spectroscopy using deuterium‐labeled NADH (4R‐2H‐NADH) and molecular docking simulations to reveal that the pro‐S hydrogen at the C4 position of the nicotinamide moiety in NADH was specifically transferred to the flavin‐N5 atom of FNM. Altogether, our findings may aid in the improvement of the indigo dyeing (Aizome) process. The stereospecificity of hydride transfer from NADH to flavin mononucleotide (FMN) in reactions catalyzed by the FMN‐dependent NADH‐indigo reductase from the thermophilic bacterium Bacillus smithii was studied. Both analyses of 1H‐NMR spectroscopy using deuterium‐labeled NADH (4R‐2H‐NADH) and molecular docking simulations showed that the pro‐S hydrogen of NADH was specifically transferred to the flavin‐N5 atom of FNM.