7,8- and 5,8-Linoleate diol synthases support the heterolytic scission of oxygen-oxygen bonds by different amide residues.
Linoleate diol synthases (LDS) are fungal dioxygenase-cytochrome P450 fusion enzymes. They oxidize 18:2n-6 sequentially to 8R-hydroperoxylinoleic acid (8R-HPODE) and 7S,8S- or 5S,8R-dihydroxylinoleic acids (DiHODE) by intramolecular oxygen transfer. The P450 domains contain a conserved sequence, Ala-Asn-Gln-Xaa-Gln, presumably located in the I-helices. The Asn938Leu replacement of 7,8-LDS of Gaeumannomyces graminis virtually abolished and the Asn938Asp and Asn938Gln replacements reduced the hydroperoxide isomerase activity. Gln941Leu and Gln941Glu substitutions had little effects. Replacements of the homologous Asn(887) and Gln(890) residues of 5,8-LDS of Aspergillus fumigatus yielded the opposite results. Asn887Leu and Asn887Gln of 5,8-LDS retained 5,8-DiHODE as the main metabolite with an increased formation of 6,8- and 8,11-DiHODE, whereas Gln890Leu almost abolished the 5,8-LDS activity. Replacement of Gln(890) with Glu also retained 5,8-DiHODE as the main product, but shifted oxygenation from C-5 to C-7 and C-11 and to formation of epoxyalcohols by homolytic scission of 8R-HPODE. P450 hydroxylases usually contain an "acid-alcohol" pair in the I-helices for the heterolytic scission of O2 and formation of compound I (Por(+) Fe(IV)=O) and water. The function of the acid-alcohol pair appears to be replaced by two different amide residues, Asn(938) of 7,8-LDS and Gln(890) of 5,8-LDS, for heterolysis of 8R-HPODE to generate compound I.
Department of Pharmaceutical Biosciences, Division of Biochemical Pharmacology, Uppsala Biomedical Center, Uppsala University, SE-75124 Uppsala, Sweden.
Inga
I
Hoffmann
Ernst H
EH
Oliw
D000577:Amides / Q000737:chemistry*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001203:Ascomycota / Q000201:enzymology; D001232:Aspergillus fumigatus / Q000201:enzymology; D019295:Computational Biology; D017124:Conserved Sequence; D008958:Models, Molecular; D010100:Oxygen / Q000737:chemistry*; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary
D000577:Amides; D010105:Oxygenases; C122014:linoleate diol synthase; D010100:Oxygen
Heme peroxidase; Mutagenesis site-specific; Oxygenation mechanism; Oxylipin biosynthesis; P450 class III; Prostacyclin synthase