title: Probing the NADH- and Methyl Red-binding site of a FMN-dependent azoreductase (AzoA) from Enterococcus faecalis.
abstract: AzoA from Enterococcus faecalis is a member of the polymeric flavin-dependent NADH-preferred azoreductase group. Little is known about the binding and interaction of NADH and azo dye in the azoreductase group. A synergetic strategy based on computational prediction, reverse genetics validation coupled with site-directed mutagenesis, and reconstruction of mutation network was used to investigate the binding and interaction of NADH and a model azo dye, Methyl Red, with AzoA. Methyl Red and NADH interacted in a unique binding mode in which the benzoic acid moiety of Methyl Red and the nicotinamide ring of NADH were not parallel to the flavin isoalloxazine ring, but lay against it at angles of ∼45° and ∼35°, respectively. The adenine ribose moiety of NADH was surrounded by loop ℓ2 on chain B and α3 on chain A in a typical Rossmann fold. There were 12 and 19 amino acid residues that could participate in the binding of Methyl Red and NADH, respectively, especially the residues Tyr-129 and Asp-184. The functional perturbation effects of 13 residues, including Tyr-129 and Asp-184, were mapped to reconstruct the mutation network, which confirmed the proposed binding modes and also provided insights into the interaction among NADH, FMN and Methyl Red.
authors.affiliation: Division of Microbiology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, United States.
authors.forename: Jinhui
authors.initials: J
authors.lastname: Feng
authors.forename: Ohgew
authors.initials: O
authors.lastname: Kweon
authors.forename: Haiyan
authors.initials: H
authors.lastname: Xu
authors.forename: Carl E
authors.initials: CE
authors.lastname: Cerniglia
authors.forename: Huizhong
authors.initials: H
authors.lastname: Chen
mesh_terms: D001391:Azo Compounds / Q000737:chemistry*; D001665:Binding Sites; D013293:Enterococcus faecalis / Q000201:enzymology*; D004789:Enzyme Activation; D005486:Flavin Mononucleotide / Q000737:chemistry*; D015336:Molecular Probe Techniques; D009243:NAD / Q000737:chemistry*; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry*; D009601:Nitroreductases; D011485:Protein Binding
chemical_list: D001391:Azo Compounds; D009243:NAD; C008492:methyl red; D005486:Flavin Mononucleotide; D009247:NADH, NADPH Oxidoreductases; D009601:Nitroreductases; C021106:azoreductase