Identification and functional analysis of new peroxygenases in oat.

MAIN CONCLUSION: Two new peroxygenases for the biosynthesis of epoxy fatty acids in oat were identified and functionally analyzed by heterologous expression along with rationally designed site-directed mutagenesis. Oat (Avena sativa L.) contains a large family of peroxygenases, a group of heme-containing monooxygenases catalyzing hydroperoxide-dependent epoxidation of unsaturated fatty acids. Here, we report identification and functional analysis of two new peroxygenases AsPXG2 and AsPXG3 from oat. The open reading frame (ORF) of AsPXG2 contains 702 bps encoding a polypeptide of 233 amino acids, while the ORF of AsPXG3 is 627 bps coding for 208 amino acids. Both AsPXG2 and AsPXG3 comprise a single transmembrane domain, conserved histidines for heme binding and a conserved EF-hand motif for calcium binding, but they only share about 50% amino acid sequence identity with each other. When expressed in Escherichia coli and Pichia pastoris, AsPXG3 showed high epoxidation activity, while AsPXG2 exhibited no activity in E. coli and low activity in P. pastoris. AsPXG3 could effectively epoxidize both mono- and polyunsaturated fatty acids with linolenic acid being the most preferred substrate. Site-directed mutagenesis was employed to investigate the structure-function relationship of oat peroxygenase on 12 conserved residues of AsPXG3. Replacement of two conserved histidines, the ligands to the prosthetic heme group of the peroxygenase, by alanine resulted in complete loss of activity. Substitution of three conserved residues surrounding the two histidines resulted in reduction of the enzymatic activity by more than 80%. These results imply that these conserved residues might be located in or near the catalytic pocket, where the two histidine residues coordinate the heme group and the surrounding residues define the shape and size of the pocket for interaction with the heme as well as two substrates.

Responses of a pair of flying locusts to lateral looming visual stimuli.

We presented a pair of locusts flying loosely tethered with laterally looming discs. Two experiments tested whether looming-evoked flight behaviour was affected by the presence (1) or relative position (2) of a conspecific. We recorded: the type of behavioural response, motion within 6 degrees of freedom, behavioural onset time and duration, distance between individuals and relative direction of motion. Response distributions of the locust furthest from the stimulus (L1) were not affected by the presence or relative position of a conspecific, whereas distributions of the closer locust (L2) were affected by its position relative to the stimulus. Motion tracks of L1 were affected by the presence of L2, which generated relatively robust responses directed forward and away from the stimulus. Translational and rotational motion of L1 differed across treatments in both experiments, whereas L2 motion was less sensitive to the presence or position of a conspecific. The start and duration of the behaviour were invariant to the presence or position of a conspecific and locust pairs maintained a fixed distance during responses to looming. Results suggest that looming-evoked behaviour is influenced by visual cues from a conspecific in the vicinity.