title: The oxygenase component of phenol hydroxylase from Acinetobacter radioresistens S13.
abstract: Phenol hydroxylase (PH) from Acinetobacter radioresistens S13 represents an example of multicomponent aromatic ring monooxygenase made up of three moieties: a reductase (PHR), an oxygenase (PHO) and a regulative component (PHI). The function of the oxygenase component (PHO), here characterized for the first time, is to bind molecular oxygen and catalyse the mono-hydroxylation of substrates (phenol, and with less efficiency, chloro- and methyl-phenol and naphthol). PHO was purified from extracts of A. radioresistens S13 cells and shown to be a dimer of 206 kDa. Each monomer is composed by three subunits: alpha (54 kDa), beta (38 kDa) and gamma (11 kDa). The gene encoding PHO alpha (named mopN) was cloned and sequenced and the corresponding amino acid sequence matched with that of functionally related oxygenases. By structural alignment with the catalytic subunits of methane monooxygenase (MMO) and alkene monooxygenase, we propose that PHO alpha contains the enzyme active site, harbouring a dinuclear iron centre Fe-O-Fe, as also suggested by spectral analysis. Conserved hydrophobic amino acids known to define the substrate recognition pocket, are also present in the alpha-subunit. The prevalence of alpha-helices (99.6%) as studied by CD confirmed the hypothized structural homologies between PHO and MMO. Three parameters (optimum ionic strength, temperature and pH) that affect kinetics of the overall phenol hydroxylase reaction were further analyzed with a fixed optimal PHR/PHI/PHO ratio of 2/1/1. The highest level of activity was evaluated between 0.075 and 0.1 m of ionic strength, the temperature dependence showed a maximum of activity at 24 degrees C and finally the pH for optimal activity was determined to be 7.5.
authors.affiliation: Dipartimento di Biologia Animale e dell'Uomo, Università di Torino, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Divari
authors.forename: Francesca
authors.initials: F
authors.lastname: Valetti
authors.forename: Patrizia
authors.initials: P
authors.lastname: Caposio
authors.forename: Enrica
authors.initials: E
authors.lastname: Pessione
authors.forename: Maria
authors.initials: M
authors.lastname: Cavaletto
authors.forename: Ersilia
authors.initials: E
authors.lastname: Griva
authors.forename: Giorgio
authors.initials: G
authors.lastname: Gribaudo
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Gilardi
authors.forename: Carlo
authors.initials: C
authors.lastname: Giunta
mesh_terms: D000150:Acinetobacter / Q000201:enzymology*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002455:Cell Division; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D016680:Genome, Bacterial; D006863:Hydrogen-Ion Concentration; D007477:Ions; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D010101:Oxygen Consumption; D010105:Oxygenases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry; D013696:Temperature; D013997:Time Factors; D014466:Ultraviolet Rays
chemical_list: D007477:Ions; D009243:NAD; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C021555:methane monooxygenase; C022336:phenol 2-monooxygenase