Critical amino acids for the 8(R)-dioxygenase activity of linoleate diol synthase. A comparison with cyclooxygenases.
7,8-Linoleate diol synthase (7,8-LDS) of the take-all fungus and cyclooxygenases can be aligned with approximately 24% amino acid identity and both form a tyrosyl radical during catalysis. 7,8-LDS was expressed in insect cells with native 8R-dioxygenase and hydroperoxide isomerase activities. We studied conserved residues of 7,8-LDS, which participate in cyclooxygenases for heme binding (His residues), hydrogen abstraction (Tyr), positioning (Tyr, Trp), and ionic binding of substrates (Arg). Site-directed mutagenesis abolished 8R-dioxygenase activities with exception of the putative distal histidine (His203Gln) and a tyrosine residue important for hydrogen bonding and substrate positioning (Tyr329Phe). The results demonstrate structural similarities between 7,8-LDS and cyclooxygenases.
Garscha
Ulrike
U
Department of Pharmaceutical Biosciences, Uppsala University, SE 75124 Uppsala, Sweden.
Oliw
Ernst H
EH
D000595:Amino Acid Sequence; D000818:Animals; D001203:Ascomycota / Q000201:enzymology*; D002384:Catalysis; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000235:genetics; D051381:Rats; D016415:Sequence Alignment; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
D005656:Fungal Proteins; D014443:Tyrosine; D006639:Histidine; D014364:Tryptophan; D010105:Oxygenases; C122014:linoleate diol synthase; D011451:Prostaglandin-Endoperoxide Synthases