| dc.description.abstracteng | In this study recombinant CYP74-enzymes AOS1, AOS2 and HPL from the moss P. patens, DES from S. tuberosum and EAS from the lancelet B. floridae were expressed in E. coli and purified for further analysis. PpAOS1 and BfEAS were biochemically characterized. Substrate specificities against hydroperoxides derived from C18 fatty acids LA and α-LeA (PpAOS1 and BfEAS), C20 fatty acid ARA (PpAOS1) as well as C22 fatty acid DHA (BfEAS) and pH optima were determined. Both enzymes showed slightly acidic pH (PpAOS1 6.5 and BfEAS 6.75). UV/vis and CD spectroscopy analysis were performed to characterize the heme content and structure of the enzymes.
Analysis of products of AOS, DES, EAS and HPL wild types showed that these enzymes have inherent side activities of the other enzymes beside their main enzyme activity. Thus PpHPL has inherent AOS activity, PpAOS1 as well as StDES have inherent HPL activity, whereas BfEAS has inherent HPL- and DES activity.
Site-directed mutagenesis was performed in order to identify amino acid residues essential for catalytic activity. Analysis of products of conversions of PpAOS1 with 9-and 13-hydroperoxides derived from LA and α-LeA showed that enzyme activity can be altered from AOS to HPL by substitution of an essential determinant (PpAOS1_F93L). This mutagenesis concept was reported before for an AOS from Arabidopsis (AtAOS) with specificity for 13-hydroperoxides (Lee et al., 2008). In the present study this concept has been extended to AOS with specificity for 9-and 13-hydroperoxide. The results showed that this concept works for both specific and unspecific enzymes. For PpHPL this concept was not applicable suggesting that for the alteration of HPL activity other determinants were essential.
Site-directed mutagenesis of StDES for product analysis generated mutants that had very low or no activity to perform the product analysis.
All purified variants of BfEAS were catalytically active. Product analysis showed that the mutational concept of AtAOS (Lee et al., 2008) can also be applied to BfEAS. It was observed that EAS activity decreased by mutating the conserved F (BfEAS_F58L) and the conserved P (BfEAS_P75A). But unlike in AtAOS or in PpAOS1 no significant increase of HPL activity was detected, because in comparison to the other formed products the amount of oxo fatty acids was nearly the same. DES activity like in the wild type was not detected in the variants. The results suggest like in PpAOS1 that the concept can be applied to unspecific enzymes. BfEAS is like PpAOS1 and unspecific enzyme.
The deletion of the 9AS-insert, which is characteristic for CYP74-enzyme, had no influence on enzyme activity. The BfEAS_9AS-deletion variant was still active.
Product analysis by RP-HPLC/MS2 confirmed the formation of epoxy alcohols by conversions of BfEAS with 9- and 13-hydroperoxides derived from LA and α-LeA. The detected fragment patterns were similar to that of wild type (Cristea and Oliw, 2006, Oliw et al., 2006).
Product analysis showed that the catalytic trajectories of PpAOS1 and PpHPL as well as BfEAS, DES and HPL are closely interconnected and can be interconverted by exchanges of special single amino acids. | de |