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<p>Abstract</p> <p>Background</p> <p>The moss <it>Physcomitrella patens</it> contains C₁₈- as well as C₂₀-polyunsaturated fatty acids that can be metabolized by different enzymes to form oxylipins such as the cyclopentenone <it>cis</it>(+)-12-oxo phytodienoic acid. Mutants defective in the biosynthesis of cyclopentenones showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis. The initial step in this biosynthetic route is the conversion of a fatty acid hydroperoxide to an allene oxide. This reaction is catalyzed by allene oxide synthase (AOS) belonging as hydroperoxide lyase (HPL) to the cytochrome P450 family Cyp74. In this study we characterized two AOS from <it>P. patens</it>, PpAOS1 and PpAOS2<it>.</it></p> <p>Results</p> <p>Our results show that PpAOS1 is highly active with both C₁₈ and C₂₀-hydroperoxy-fatty acid substrates, whereas PpAOS2 is fully active only with C₂₀-substrates, exhibiting trace activity (~1000-fold lower k_cat/K_M) with C₁₈ substrates. Analysis of products of PpAOS1 and PpHPL further demonstrated that both enzymes have an inherent side activity mirroring the close inter-connection of AOS and HPL catalysis. By employing site directed mutagenesis we provide evidence that single amino acid residues in the active site are also determining the catalytic activity of a 9-/13-AOS – a finding that previously has only been reported for substrate specific 13-AOS. However, PpHPL cannot be converted into an AOS by exchanging the same determinant. Localization studies using YFP-labeled AOS showed that PpAOS2 is localized in the plastid while PpAOS1 may be found in the cytosol. Analysis of the wound-induced <it>cis</it>(+)-12-oxo phytodienoic acid accumulation in <it>PpAOS1</it> and <it>PpAOS2</it> single knock-out mutants showed that disruption of PpAOS1, in contrast to PpAOS2, results in a significantly decreased <it>cis</it>(+)-12-oxo phytodienoic acid formation. However, the knock-out mutants of neither <it>PpAOS1</it> nor <it>PpAOS2</it> showed reduced fertility, aberrant sporophyte morphology or interrupted sporogenesis.</p> <p>Conclusions</p> <p>Our study highlights five findings regarding the oxylipin metabolism in <it>P. patens</it>: (i) Both AOS isoforms are capable of metabolizing C₁₈- and C₂₀-derived substrates with different specificities suggesting that both enzymes might have different functions. (ii) Site directed mutagenesis demonstrated that the catalytic trajectories of 9-/13-PpAOS1 and PpHPL are closely inter-connected and PpAOS1 can be inter-converted by a single amino acid exchange into a HPL. (iii) In contrast to PpAOS1, PpAOS2 is localized in the plastid where oxylipin metabolism takes place. (iv) PpAOS1 is essential for wound-induced accumulation of <it>cis</it>(+)-12-oxo phytodienoic acid while PpAOS2 appears not to be involved in the process. (v) Knock-out mutants of neither AOS showed a deviating morphological phenotype suggesting that there are overlapping functions with other Cyp74 enzymes.</p>