The oxygenase component of phenol hydroxylase from Acinetobacter radioresistens S13

Sara Divari, Francesca Valetti, Patrizia Caposio, Enrica Pessione, Maria Cavaletto, Ersilia Griva, Giorgio Gribaudo, Gianfranco Gilardi, Carlo Giunta

Research output: Contribution to journalArticlepeer-review

35 Scopus citations


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: α (54 kDa), β (38 kDa) and γ (11 kDa). The gene encoding PHO α (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 α 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 α-subunit. The prevalence of α-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°C and finally the pH for optimal activity was determined to be 7.5.

Original languageEnglish (US)
Pages (from-to)2244-2253
Number of pages10
JournalEuropean Journal of Biochemistry
Issue number10
StatePublished - May 2003
Externally publishedYes


  • Catalytic subunit
  • Molecular cloning
  • Multicomponent monooxygenase
  • Phenol hydroxylase
  • Purification

ASJC Scopus subject areas

  • Biochemistry


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