Cloning of linoleate diol synthase reveals homology with prostaglandin H synthases.
Linoleate diol synthase is a homotetrameric ferric hemeprotein, which catalyzes dioxygenation of linoleic acid to (8R)-hydroperoxylinoleate and isomerization of the hydroperoxide to (7S,8S)-dihydroxylinoleate. Ferryl intermediates and a tyrosyl radical are formed in the reaction. Linoleate diol synthase was digested with endoproteinase Lys-C, and internal peptides were sequenced. The sequence information was used for reverse transcription-polymerase chain reaction analysis, and a cDNA probe was obtained. Northern blot analysis of linoleate diol synthase suggested a 3.7-kilobase pair (kb) mRNA. A full-length clone of the linoleate diol synthase gene was obtained by screening of a genomic lambda-ZAP II library of the fungus Gaeumannomyces graminis. The 5'-untranslated region contained CAAT- and TATA-like boxes. The gene contained three short introns and spanned over 3.2-kb. The deduced open reading frame consisted of 2.9-kb, which corresponded to 978 amino acids and a molecular subunit mass of 108,000. Data base analysis with the gapped BLAST algorithm showed that 391 residues of linoleate diol synthase was 23-24% identical and 36-37% positive with the catalytic domain of mammalian prostaglandin H (PGH) synthase-2. Based on homology with PGH synthases, the proximal heme ligand of linoleate diol synthase was tentatively identified as His-379 and the important tyrosine for catalysis as residue 376 (apparent consensus EFNXXXYXWH). The distal heme ligand was tentatively identified as His-203 (apparent consensus THXXFXT). We conclude from catalytic and structural similarities that linoleate diol synthase and PGH synthases likely share common ancestry and may belong to a gene family of fatty acid heme dioxygenases.
Hörnsten
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Department of Pharmaceutical Biosciences, Uppsala Biomedical Center, SE-751 24 Uppsala, Sweden.
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Osbourn
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Garosi
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Hellman
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Wernstedt
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Oliw
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics*; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D010105:Oxygenases; C122014:linoleate diol synthase; D011451:Prostaglandin-Endoperoxide Synthases