PpoC from Aspergillus nidulans is a fusion protein with only one active haem.
In Aspergillus nidulans Ppos [psi (precocious sexual inducer)-producing oxygenases] are required for the production of so-called psi factors, compounds that control the 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 haem-containing domains are predicted: a fatty acid haem peroxidase/dioxygenase domain in the N-terminal region and a P450 haem-thiolate domain in the C-terminal region. Whereas PpoA was shown to use both haem domains for its bifunctional catalytic activity (linoleic acid 8-dioxygenation and 8-hydroperoxide isomerization), we found that PpoC apparently only harbours a functional haem peroxidase/dioxygenase domain. Consequently, we observed that PpoC catalyses mainly the dioxygenation of linoleic acid (18:2Delta9Z,12Z), yielding 10-HPODE (10-hydroperoxyoctadecadienoic acid). No isomerase activity was detected. Additionally, 10-HPODE was converted at lower rates into 10-KODE (10-keto-octadecadienoic acid) and 10-HODE (10-hydroxyoctadecadienoic acid). In parallel, decomposition of 10-HPODE into 10-ODA (10-octadecynoic acid) and volatile C-8 alcohols that are, among other things, responsible for the characteristic mushroom flavour. Besides these principle differences we also found that PpoA and PpoC can convert 8-HPODE and 10-HPODE into the respective epoxy alcohols: 12,13-epoxy-8-HOME (where HOME is hydroxyoctadecenoic acid) and 12,13-epoxy-10-HOME. By using site-directed mutagenesis we demonstrated that both enzymes share a similar mechanism for the oxidation of 18:2Delta9Z,12Z; they both use a conserved tyrosine residue 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.
Brodhun
Florian
F
Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany.
Schneider
Stefan
S
Göbel
Cornelia
C
Hornung
Ellen
E
Feussner
Ivo
I
D000438:Alcohols / Q000737:chemistry; D001233:Aspergillus nidulans / Q000378:metabolism*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics; D005227:Fatty Acids / Q000737:chemistry; D005656:Fungal Proteins / Q000378:metabolism*; D016681:Genome, Fungal; D006418:Heme / Q000737:chemistry*; D007700:Kinetics; D019787:Linoleic Acid / Q000737:chemistry; D008041:Linoleic Acids / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D054883:Oxylipins / Q000737:chemistry; D017434:Protein Structure, Tertiary
D000438:Alcohols; D005227:Fatty Acids; D005656:Fungal Proteins; D008041:Linoleic Acids; D054883:Oxylipins; D006418:Heme; C058335:10-hydroperoxy-8,12-octadecadienoic acid; D019787:Linoleic Acid; D049308:Dioxygenases