Probing the NADH- and Methyl Red-binding site of a FMN-dependent azoreductase (AzoA) from Enterococcus faecalis

► A synergetic strategy was used to investigate the binding of NADH and Methyl Red in AzoA. ► Methyl Red and NADH lay against the flavin isoalloxazine ring at an angle of 45° and 35°. ► There were 12 and 19 amino acid residues participated in the binding of Methyl Red and NADH. ► Functional perturbation effects of the 13 residues were mapped to reconstruct the mutation network.