PpoC from Aspergillus nidulans is a Fusion Protein with one active Heme

International audienceIn Aspergillus nidulans psi factor producing oxygenases (Ppos) are required for the production of so-called psi factors (precocious sexual inducer) - compounds that control balance between the sexual and asexual life cycle of the fungus. The genome of A. nidulans harbours three different ppo genes: ppoA, ppoB and ppoC. For all three enzymes two different heme containing domains are predicted: a fatty acid heme peroxidase/dioxygenase domain for the N-terminal region and a P450 heme thiolate domain for the C-terminal region. While PpoA was shown to use both heme domains for its bifunctional catalytic activity (linoleic acid 8-dioxygenation and 8-hydroperoxide isomerisation), we found that PpoC apparently only harbours a functional heme peroxidase/dioxygenase domain. Consequently, we observed that PpoC catalyzes mainly the dioxygenation of linoleic acid (18:2Δ9Z,12Z), yielding 10-hydroperoxy linoleic acid (10-HPODE). No isomerase activity was detected. Additionally, 10-HPODE was converted at lower rates into 10-keto linoleic acid (10-KODE) and 10-hydroxy linoleic acid (10-HODE). In parallel, decomposition of 10-HPODE into 10-oxo decenoic acid (10-ODA) and volatile C8 alcohols that are inter alia responsible for the characteristic mushroom flavour. Beside these principle differences we also found that PpoA and PpoC can convert 8-HPODE and 10-HPODE to the respective epoxy alcohols: 12,13-Epoxy-8-hydroxy octadecenoic acid and 12,13-epoxy-10-hydroxy octadecenoic acid. By using site directed mutagenesis we could demonstrate that both enzymes share a similar mechanism for the oxidation of 18:2Δ9Z,12Z: They both use a conserved tyrosine for catalysis and the directed oxygenation at the C-8 and C-10 is most likely controlled by conserved valine/leucine residues in the dioxygenase domain