title: Phenol hydroxylase from Acinetobacter radioresistens S13. Isolation and characterization of the regulatory component.
abstract: This paper reports the isolation and characterization of the regulatory moiety of the multicomponent enzyme phenol hydroxylase from Acinetobacter radioresistens S13 grown on phenol as the only carbon and energy source. The whole enzyme comprises an oxygenase moiety (PHO), a reductase moiety (PHR) and a regulatory moiety (PHI). PHR contains one FAD and one iron-sulfur cluster, whose function is electron transfer from NADH to the dinuclear iron centre of the oxygenase. PHI is required for catalysis of the conversion of phenol to catechol in vitro, but is not required for PHR activity towards alternative electron acceptors such as cytochrome c and Nitro Blue Tetrazolium. The molecular mass of PHI was determined to be 10 kDa by SDS/PAGE, 8.8 kDa by MALDI-TOF spectrometry and 18 kDa by gel-permeation. This finding suggests that the protein in its native state is a homodimer. The isoelectric point is 4.1. PHI does not contain any redox cofactor and does not bind ANS, a fluorescent probe for hydrophobic sites. The N-terminal sequence is similar to those of the regulatory proteins of phenol hydroxylase from A. calcoaceticus and Pseudomonas CF 600. In the reconstituted system, optimal reaction rate was achieved when the stoichiometry of the components was 2 PHR monomers: 1 PHI dimer: 1 PHO (alphabetagamma) dimer. PHI interacts specifically with PHR, promoting the enhancement of FAD fluorescence emission. This signal is diagnostic of a conformational change of PHR that might result in a better alignment with respect to PHO.
authors.affiliation: Dipartimento di Biologia Animale e dell'Uomo, Università di Torino, Italy.
authors.forename: Ersilia
authors.initials: E
authors.lastname: Griva
authors.forename: Enrica
authors.initials: E
authors.lastname: Pessione
authors.forename: Sara
authors.initials: S
authors.lastname: Divari
authors.forename: Francesca
authors.initials: F
authors.lastname: Valetti
authors.forename: Maria
authors.initials: M
authors.lastname: Cavaletto
authors.forename: Gian Luigi
authors.initials: GL
authors.lastname: Rossi
authors.forename: Carlo
authors.initials: C
authors.lastname: Giunta
mesh_terms: D000150:Acinetobacter / Q000201:enzymology*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000502:physiology; D004795:Enzyme Stability / Q000502:physiology; D005456:Fluorescent Dyes; D007526:Isoelectric Point; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010084:Oxidation-Reduction; D019800:Phenol / Q000378:metabolism; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D009097:Multienzyme Complexes; D021122:Protein Subunits; D019800:Phenol; D006899:Mixed Function Oxygenases; C022336:phenol 2-monooxygenase