title: Biochemical characterization of the oxygenation of unsaturated fatty acids by the dioxygenase and hydroperoxide isomerase of Pseudomonas aeruginosa 42A2.
abstract: We have studied oxygenation of fatty acids by cell extract of Pseudomonas aeruginosa 42A2. Oleic acid ((9Z)-18:1) was transformed to (10S)-hydroperoxy-(8E)-octadecenoic acid ((10S)-HPOME) and to (7S,10S)-dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Experiments under oxygen-18 showed that 7,10-DiHOME contained oxygen from air and was formed sequentially from (10S)-HPOME by isomerization. (10R)-HPOME was not isomerized. The (10S)-dioxygenase and hydroperoxide isomerase activities co-eluted on ion exchange chromatography and on gel filtration with an apparent molecular size of approximately 50 kDa. 16:1n-7, 18:2n-6, and 20:1n-11 were also oxygenated to 7,10-dihydroxy fatty acids, and (8Z)-18:1 was oxygenated to 6,9-dihydroxy-(7E)-octadecenoic acid. A series of fatty acids with the double bond positioned closer to ((6Z)-18:1, (5Z,9Z)-18:2) or more distant from the carboxyl group ((11Z)-, (13Z)-, and (15Z)-18:1) were poor substrates. The oxygenation mechanism was studied with [7S-(2)H]18:1n-9, [7R-(2)H]18:2n-6, and [8R-(2)H]18:2n-6 as substrates. The pro-R hydrogen at C-8 was lost in the biosynthesis of (10S)-HPODE, whereas the pro-S hydrogen was lost and the pro-R hydrogen was retained at C-7 during biosynthesis of the 7,10-dihydroxy metabolites. Analysis of the fatty acid composition of P. aeruginosa revealed relatively large amounts of (9E/Z)-16:1 and (11E/Z)-18:1 and only traces of 18:1n-9. We found that (11Z)-18:1 (vaccenic acid) was transformed to (11S,14S)-dihydroxy-(12E)-octadecenoic acid and to a mixture of 11- and 12-HPOME, possibly due to reverse orientation of (11Z)-18:1 at the active site compared with oleic acid. The reaction mechanism of the hydroperoxide isomerase suggests catalytic similarities to cytochrome P450.
authors.affiliation: Division of Biochemical Pharmacology, Department of Pharmaceutical Bioscience, Uppsala Biomedical Center, P.O. Box 591, SE-751 24 Uppsala, Sweden; Laboratori de Microbiologia, Facultat de Farmàcia, Barcelona E-08028, Spain.
authors.forename: Eriel
authors.initials: E
authors.lastname: Martínez
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Hamberg
authors.affiliation: Departament de Bioquimica i Biologia Molecular, Universitat de Barcelona, Barcelona E-08028, Spain.
authors.forename: Montse
authors.initials: M
authors.lastname: Busquets
authors.affiliation: Laboratori de Microbiologia, Facultat de Biologia, Barcelona E-08028, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Díaz
authors.affiliation: Laboratori de Microbiologia, Facultat de Farmàcia, Barcelona E-08028, Spain.
authors.forename: Angeles
authors.initials: A
authors.lastname: Manresa
authors.affiliation: Division of Biochemical Pharmacology, Department of Pharmaceutical Bioscience, Uppsala Biomedical Center, P.O. Box 591, SE-751 24 Uppsala, Sweden. Electronic address: Ernst.Oliw@farmbio.uu.se.
authors.forename: Ernst H
authors.initials: EH
authors.lastname: Oliw
mesh_terms: D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D049308:Dioxygenases / Q000378:metabolism*; D005227:Fatty Acids / Q000737:chemistry; D005231:Fatty Acids, Unsaturated / Q000737:chemistry*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006836:Hydro-Lyases / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005227:Fatty Acids; D005231:Fatty Acids, Unsaturated; D049308:Dioxygenases; D006836:Hydro-Lyases; C057374:hydroperoxide dehydratase; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase; D010100:Oxygen