title: Leucine/valine residues direct oxygenation of linoleic acid by (10R)- and (8R)-dioxygenases: expression and site-directed mutagenesis oF (10R)-dioxygenase with epoxyalcohol synthase activity.
abstract: Linoleate (10R)-dioxygenase (10R-DOX) of Aspergillus fumigatus was cloned and expressed in insect cells. Recombinant 10R-DOX oxidized 18:2n-6 to (10R)-hydroperoxy-8(E),12(Z)-octadecadienoic acid (10R-HPODE; approximately 90%), (8R)-hydroperoxylinoleic acid (8R-HPODE; approximately 10%), and small amounts of 12S(13R)-epoxy-(10R)-hydroxy-(8E)-octadecenoic acid. We investigated the oxygenation of 18:2n-6 at C-10 and C-8 by site-directed mutagenesis of 10R-DOX and 7,8-linoleate diol synthase (7,8-LDS), which forms approximately 98% 8R-HPODE and approximately 2% 10R-HPODE. The 10R-DOX and 7,8-LDS sequences differ in homologous positions of the presumed dioxygenation sites (Leu-384/Val-330 and Val-388/Leu-334, respectively) and at the distal site of the heme (Leu-306/Val-256). Leu-384/Val-330 influenced oxygenation, as L384V and L384A of 10R-DOX elevated the biosynthesis of 8-HPODE to 22 and 54%, respectively, as measured by liquid chromatography-tandem mass spectrometry analysis. The stereospecificity was also decreased, as L384A formed the R and S isomers of 10-HPODE and 8-HPODE in a 3:2 ratio. Residues in this position also influenced oxygenation by 7,8-LDS, as its V330L mutant augmented the formation of 10R-HPODE 3-fold. Replacement of Val-388 in 10R-DOX with leucine and phenylalanine increased the formation of 8R-HPODE to 16 and 36%, respectively, whereas L334V of 7,8-LDS was inactive. Mutation of Leu-306 with valine or alanine had little influence on the epoxyalcohol synthase activity. Our results suggest that Leu-384 and Val-388 of 10R-DOX control oxygenation of 18:2n-6 at C-10 and C-8, respectively. The two homologous positions of prostaglandin H synthase-1, Val-349 and Ser-353, are also critical for the position and stereospecificity of the cyclooxygenase reaction.
authors.affiliation: Department of Pharmaceutical Biosciences, Uppsala Biomedical Center, SE-751 24 Uppsala, Sweden.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Garscha
authors.affiliation: Department of Pharmaceutical Biosciences, Uppsala Biomedical Center, SE-751 24 Uppsala, Sweden. Electronic address: Ernst.Oliw@farmbio.uu.se.
authors.forename: Ernst H
authors.initials: EH
authors.lastname: Oliw
mesh_terms: D000818:Animals; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D002460:Cell Line; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D007313:Insecta; D019746:Intramolecular Oxidoreductases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007930:Leucine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008025:Ligases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D019787:Linoleic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008041:Linoleic Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014633:Valine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D008041:Linoleic Acids; D011994:Recombinant Proteins; C075807:8-hydroperoxylinoleic acid; D019787:Linoleic Acid; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase; D008025:Ligases; D007930:Leucine; D014633:Valine