title: OnpA, an unusual flavin-dependent monooxygenase containing a cytochrome b(5) domain.
abstract: ortho-Nitrophenol 2-monooxygenase (EC 1.14.13.31) from Alcaligenes sp. strain NyZ215 catalyzes monooxygenation of ortho-nitrophenol to form catechol via ortho-benzoquinone. Sequence analysis of this onpA-encoded enzyme revealed that it contained a flavin-binding monooxygenase domain and a heme-binding cytochrome b(5) domain. OnpA was purified to homogeneity as a His-tagged protein and was considered a monomer, as determined by gel filtration. FAD and heme were identified by high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry (HPLC-MS) as cofactors in this enzyme, and quantitative analysis indicated that 1 mol of the purified recombinant OnpA contained 0.66 mol of FAD and 0.20 mol of heme. However, the enzyme activity of OnpA was increased by 60% and 450% after addition of FAD and hemin, respectively, suggesting that the optimal stoichiometry was 1:1:1. In addition, site-directed mutagenesis experiments confirmed that two highly conserved histidines located in the cytochrome b(5) domain were associated with binding of the heme, and the cytochrome b(5) domain was involved in the OnpA activity. These results indicate that OnpA is an unusual FAD-dependent monooxygenase containing a fused cytochrome b(5) domain that is essential for its activity. Therefore, we here demonstrate a link between cytochrome b(5) and flavin-dependent monooxygenases.
authors.affiliation: Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Xiao
authors.forename: Ting-Ting
authors.initials: TT
authors.lastname: Liu
authors.forename: Hui
authors.initials: H
authors.lastname: Dai
authors.forename: Jun-Jie
authors.initials: JJ
authors.lastname: Zhang
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.forename: Huiru
authors.initials: H
authors.lastname: Tang
authors.forename: David J
authors.initials: DJ
authors.lastname: Leak
authors.forename: Ning-Yi
authors.initials: NY
authors.lastname: Zhou
mesh_terms: D000421:Alcaligenes / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003067:Coenzymes / Q000378:metabolism; D015786:Cytochromes b5 / Q000235:genetics*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D006427:Hemin / Q000378:metabolism; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
chemical_list: D003067:Coenzymes; D005182:Flavin-Adenine Dinucleotide; D006427:Hemin; D015786:Cytochromes b5; D006899:Mixed Function Oxygenases