Cloning of linoleate diol synthase reveals homology with prostaglandin H synthases

Linoleate diol synthase is a homotetrameric ferric hemeprotein, which catal yzes dioxygenation of linoleic acid to (8R)-hydroperoxylinoleate and isomer ization of the hydroperoxide to (7S,8S) dihydroxylinoleate. Ferryl intermed iates and a tyrosyl radical are formed in the reaction. Linoleate diol synt hase was digested with endoproteinase Lys-C, and internal peptides were seq uenced. The sequence information was used for reverse transcription-polymer ase chain reaction analysis, and a cDNA probe was obtained. Northern blot a nalysis 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 sc reening of a genomic A-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 rea ding 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 BLAS T algorithm showed that 391 residues of linoleate diol synthase was 23-24% identical and 36-37% positive with the catalytic domain of mammalian prosta glandin H (PGH) synthase-a. Based on homology with PGH synthases, the proxi mal heme ligand of linoleate diol synthase was tentatively identified as Hi s-379 and the important tyrosine for catalysis as residue 376 (apparent con sensus EFNXXXYXWTH). The distal heme ligand was tentatively identified as H is-203 (apparent consensus THXPFXT). We conclude from catalytic and structu ral similarities that linoleate diol synthase and PGH synthases likely shar e common ancestry and may belong to a gene family of fatty acid heme dioxyg enases.