title: Replacement of two amino acids of 9R-dioxygenase-allene oxide synthase of Aspergillus niger inverts the chirality of the hydroperoxide and the allene oxide.
abstract: The genome of Aspergillus niger codes for a fusion protein (EHA25900), which can be aligned with ~50% sequence identity to 9S-dioxygenase (DOX)-allene oxide synthase (AOS) of Fusarium oxysporum, homologues of the Fusarium and Colletotrichum complexes and with over 62% sequence identity to homologues of Aspergilli, including (DOX)-9R-AOS of Aspergillus terreus. The aims were to characterize the enzymatic activities of EHA25900 and to identify crucial amino acids for the stereospecificity. Recombinant EHA25900 oxidized 18:2n-6 sequentially to 9R-hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HPODE) and to a 9R(10)-allene oxide. 9S- and 9R-DOX-AOS catalyze abstraction of the pro-R hydrogen at C-11, but the direction of oxygen insertion differs. A comparison between twelve 9-DOX domains of 9S- and 9R-DOX-AOS revealed conserved amino acid differences, which could contribute to the chirality of products. The Gly616Ile replacement of 9R-DOX-AOS (A. niger) increased the biosynthesis of 9S-HPODE and the 9S(10)-allene oxide, whereas the Phe627Leu replacement led to biosynthesis of 9S-HPODE and the 9S(10)-allene oxide as main products. The double mutant (Gly616Ile, Phe627Leu) formed over 90% of the 9S stereoisomer of HPODE. 9S-HPODE was formed by antarafacial hydrogen abstraction and oxygen insertion, i.e., the original H-abstraction was retained but the product chirality was altered. We conclude that 9R-DOX-AOS can be altered to 9S-DOX-AOS by replacement of two amino acids (Gly616Ile, Phe627Leu) in the DOX domain.
authors.affiliation: Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden.
authors.forename: Linda
authors.initials: L
authors.lastname: Sooman
authors.affiliation: Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden.
authors.forename: Anneli
authors.initials: A
authors.lastname: Wennman
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Solna, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Hamberg
authors.affiliation: Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden.
authors.forename: Inga
authors.initials: I
authors.lastname: Hoffmann
authors.affiliation: Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, 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: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001230:Aspergillus / Q000235:genetics / Q000378:metabolism; D001234:Aspergillus niger / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D017124:Conserved Sequence; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005670:Fusarium / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006861:Hydrogen Peroxide; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008041:Linoleic Acids / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism
chemical_list: D005656:Fungal Proteins; D008041:Linoleic Acids; D011994:Recombinant Proteins; C049606:9-hydroperoxy-11,12-octadecadienoic acid; D006861:Hydrogen Peroxide; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase
keywords: Class III cytochrome P450; Fatty acid oxidation; Heme peroxidase; Linoleate diol synthase; Mass spectrometry; Site-specific mutagenesis