title: Site-directed mutagenesis of substrate binding sites of azoreductase from Rhodobacter sphaeroides.
abstract: Comparison of three-dimensional structures of flavin-dependent azoreductases revealed two conserved loops around the flavin mononucleotide (FMN) cofactor. Tyr74, His75 and Lys109 in the two loops of azoreductase AZR from Rhodobacter sphaeroides were replaced with Trp, Asn and Ala/His by site-directed mutagenesis, respectively. The optimal pH values of K109H and H75N were pH 6, and those of K109A and Y74W were pH 9. The optimal temperature (30 degrees C) was not affected by mutation. Positively charged residues at position 109 is critical for the binding of methyl red. K109 might only be involved in the binding of the 2'-phosphate group of NADPH and have no effect on the binding of NADH. Y74W and H75N mutations decreased the binding of methyl red/nitrofurazone and had no affect on the binding of NADPH.
authors.affiliation: School of Environmental and Biological Science and Technology, Dalian University of Technology, 116024, Dalian, P.R. China.
authors.forename: Guangfei
authors.initials: G
authors.lastname: Liu
authors.forename: Jiti
authors.initials: J
authors.lastname: Zhou
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.forename: Bin
authors.initials: B
authors.lastname: Yan
authors.forename: Jingmei
authors.initials: J
authors.lastname: Li
authors.forename: Hong
authors.initials: H
authors.lastname: Lu
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Qu
authors.forename: Ruofei
authors.initials: R
authors.lastname: Jin
mesh_terms: D000595:Amino Acid Sequence; D001391:Azo Compounds / Q000378:metabolism; D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009249:NADP / Q000378:metabolism; D009601:Nitroreductases; D010957:Plasmids; D017434:Protein Structure, Tertiary; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013696:Temperature
chemical_list: C469409:Acid red 14; D001391:Azo Compounds; D009249:NADP; C008492:methyl red; D009247:NADH, NADPH Oxidoreductases; D009601:Nitroreductases; C021106:azoreductase