| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 1925800 | Archives of Biochemistry and Biophysics | 2011 | 7 Pages |
5,8-Linoleate diol synthase (5,8-LDS) of Aspergillus fumigatus was cloned, expressed, and compared with 7,8-LDS of the Take-all fungus. Replacements of Tyr and Cys in the conserved YRWH and FXXGPHXCLG sequences abolished 8R-dioxygenase (8-DOX) and hydroperoxide isomerase activities, respectively. The predicted α-helices of LDS were aligned with α-helices of cyclooxygenase-1 (COX-1) to identify the 8-DOX domains. N-terminal expression constructs of 5,8- and 7,8-LDS (674 of 1079, and 673 of 1165 residues), containing one additional α-helix compared to cyclooxygenase-1, yielded prominent 8R-DOX activities with apparently unchanged or slightly lower substrate affinities, respectively. Val-328 of 5,8-LDS did not influence the position of oxygenation in contrast to the homologous residues Val-349 of COX-1 and Leu-384 of 10R-dioxygenase. We conclude that ∼675 amino acids are sufficient to support 8-DOX activity.
Research highlights► 5,8-Linoleate diol synthase (5,8-LDS) was cloned from the human pathogen, Aspergillus fumigatus. ► 5,8-LDS was expressed and compared to cyclooxygenase and 7,8-LDS. ► The dioxygenase domains of 5,8-LDS and 7,8-LDS could be expressed independently. ► The dioxygenase domains contained one more α-helix than cyclooxygenase. ► Critical amino acids for dioxygenation were conserved in LDS and cyclooxygenase.
