7,8-Linoleate diol synthase (7,8-LDS) of the take-all pathogen of wheat, Gaeumannomyces graminis, converts linoleic acid to 8R-hydroperoxyoctadecadienoic acid (8-HPODE) by 8-dioxygenase activity (8-DOX), and further isomerizes the hydroperoxide to 7S,8S-dihydroxyoctadecadienoic acid (7,8-DiHODE) by hydroperoxide isomerase activity. Sequence alignment showed homology to prostaglandin H synthase (PGHS), and both enzymes share structural and catalytic properties. The 8-DOX of 7,8-LDS was successfully expressed in Pichia pastoris and in insect cells (Sf21). Site-directed mutagenesis confirmed His379 as the proximal heme ligand and Tyr376 as a residue, which forms a tyrosyl radical and initiates catalysis. Furthermore, mutagenesis suggested His203 could be the proposed distal histidine, and Tyr329 of catalytic relevance for substrate positioning at the active site. Aspergilli are ubiquitous environmental fungi. Some species, in particular Aspergillus fumigatus, are responsible for invasive aspergillosis, which is a life-threatening disease for immunocompromised patients. A. fumigatus and A. nidulans metabolized linoleic acid to 8R-HPODE, 10R-hydroperoxyoctadecadienoic acid (10R-HPODE), 5S,8R-dihydroxyoctadecadienoic acid, and 8R,11S-dihydroxyoctadecadienoic acid. When the genomes of certain Aspergilli strains were published, several species showed at least three homologous genes (ppoA, ppoB, ppoC- psi producing oxygenases) to 7,8-LDS and PGHS. Gene deletion identified PpoA as an enzyme with 8-DOX and 5,8-hydroperoxide isomerase activities, designated 5,8-LDS in homology to 7,8-LDS. In the same way, PpoC was identified as a 10-dioxygenase (10-DOX), which converts linoleic acid to 10R-HPODE. 10-DOX differs from LDS, since it dioxygenates linoleic acid at C-10, after hydrogen abstraction at C-8 and double bond migration. 10-DOX was cloned and expressed in insect cells. Leu384 and Val388 were found to be critical for dioxygenation at C-10. Mutation to the homologous residues of 5,8- and 7,8-LDS (Leu384Val, Val388Leu) increased oxygen insertion at C-8. LDS and 10-DOX are fusion proteins with a dioxygenase and a hydroperoxide isomerase (cytochrome P450) domain with a cysteine heme ligand. The P450 domain of 10-DOX lacked the crucial cysteine heme ligand and was without hydroperoxide isomerase activity. LDSs and 10-DOX are newly characterized heme containing fungal dioxygenases, with homology to PGHS of vertebrates. Their metabolites regulate reproduction, development, and act as signal molecules with the host after pathogen attack.

The dioxygenase-cytochrome P450 fusion proteins (DOX-CYP) comprise a heme-containing enzyme family that shares structural and catalytic properties with mammalian prostaglandin H (PGH) synthases. 7,8-Linoleate diol synthase (7,8-LDS) of Gaeumannomyces graminis was first characterized, and DOX-CYP enzymes are of mechanistic and biological interest. The growing number of fungal genome sequences has revealed DOX-CYP homologues in medically and economically important species. The aim of this thesis was to identify novel members of the DOX-CYP fusion protein family. The devastating rice pathogen Magnaporthe oryzae contains two DOX-CYP genes. The fungus synthesizes 7S,8S-dihydroxyoctadecadienoic acid (7,8-DiHODE) by dioxygenation of linoleic acid to 8R-hydroperoxyoctadecadienoic acid (8R-HPODE), and subsequent isomerisation to the diol. 7,8-LDS of M. oryzae was identified by gene deletion, but the infection and reproduction processes of the Δ7,8-LDS strain were not altered. A mutant with constitutive protein kinase A activity profoundly changed the oxygenation profile, possibly due to post-translational modification. The human pathogens Aspergillus fumigatus and A. clavatus contain three DOX-CYP, designated psi producing oxygenase A (ppoA), ppoB, and ppoC, and form three oxylipins: 5S,8R-DiHODE, 8R,11S-DiHODE, and 10R-hydroxyoctadecadienoic acid.  PpoA was identified as 5,8-LDS, and ppoC as 10R-DOX. The 8,11-linoleate hydroperoxide isomerase activity was reduced by two imidazole-containing P450 inhibitors, miconazole and 1-benzylimidazole. PpoB could not be linked to the biosynthesis of 8,11-DiHODE for the following reasons: First, the 8,11-hydroperoxide isomerase activity was retained in A. fumigatus ΔppoB strains. Second, the P450 domain of the deduced ppoB of A. clavatus lacks a heme-thiolate cysteine ligand, presumably essential for hydroperoxide isomerase activity. Linoleate 9R-DOX activities of Aspergillus terreus and Lasiodiplodia theobromae were discovered. 9R-HPODE was further converted into unstable allene oxides, as judged by the accumulation of their hydrolysis products, α- and γ-ketols. These allene oxide synthase activities were specific for 9R-hydroperoxides. The 9R-DOX and AOS were found to have unique characteristics. In conclusion, novel DOX-CYP enzymes were identified in human and plant pathogenic fungi. These enzymes might be involved in biological processes, and show interesting catalytic similarities to human PGH synthase and thromboxane synthase (CYP5A).