Biochemical characterization of the oxygenation of unsaturated fatty acids by the dioxygenase and hydroperoxide isomerase of Pseudomonas aeruginosa 42A2.
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.
Martínez
Eriel
E
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.
Hamberg
Mats
M
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Busquets
Montse
M
Departament de Bioquimica i Biologia Molecular, Universitat de Barcelona, Barcelona E-08028, Spain.
Díaz
Pilar
P
Laboratori de Microbiologia, Facultat de Biologia, Barcelona E-08028, Spain.
Manresa
Angeles
A
Laboratori de Microbiologia, Facultat de Farmàcia, Barcelona E-08028, Spain.
Oliw
Ernst H
EH
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.
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
D005227:Fatty Acids; D005231:Fatty Acids, Unsaturated; D049308:Dioxygenases; D006836:Hydro-Lyases; C057374:hydroperoxide dehydratase; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase; D010100:Oxygen