Expression of 5,8-LDS of Aspergillus fumigatus and its dioxygenase domain. A comparison with 7,8-LDS, 10-dioxygenase, and cyclooxygenase.
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.
Section of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Center, SE-75124 Uppsala, Sweden.
Inga
I
Hoffmann
Fredrik
F
Jernerén
Ulrike
U
Garscha
Ernst H
EH
Oliw
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics*; D051545:Cyclooxygenase 1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D049308:Dioxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D005656:Fungal Proteins; D011994:Recombinant Proteins; D010105:Oxygenases; D049308:Dioxygenases; C122014:linoleate diol synthase; D051545:Cyclooxygenase 1