Pichia expression and mutagenesis of 7,8-linoleate diol synthase change the dioxygenase and hydroperoxide isomerase.
Linoleate diol synthases (LDS) are homologous 8(R)-dioxygenases with hydroperoxide isomerase activities, expressed in fungal pathogens of humanitarian importance. We report for the first time expression and site-directed mutagenesis of LDS. 7,8-LDS of the take-all fungus, expressed in Pichia pastoris, oxygenated 18:2n-6 to 8(R)-hydroperoxylinoleic acid, which was unexpectedly isomerized to 5,8(R)-dihydroxylinoleic acid (60% 5S) and to 8(R),13-dihydroxyoctadeca-9(E),11(E)-dienoic acid. The latter was likely formed via hydrolysis of an unstable intermediate, 8(R),9(S)-epoxyoctadeca-10(E),12(Z)-dienoic acid. A tyrosyl radical is formed during 7,8-LDS catalysis, and Tyr376 is the sequence homolog to Tyr385 of cyclooxygenase-1. Tyr376Phe retained hydroperoxide isomerase activity but lacked 8(R)-dioxygenase activity. The putative proximal heme ligand His379 and the N-glycosylation site at Asn216 appeared to be critical for 8(R)-dioxygenase activity, as His379Gln and Asn216Gln were inactive. Treatment with alpha-mannosidase to shorten N- and O-linked mannosides inhibited the hydroperoxide isomerase but not the 8(R)-dioxygenase. Our results suggest that post-translational modifications may influence the oxidation mechanism of 7,8-LDS.
Garscha
Ulrike
U
Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Biomedicum, Uppsala University, P.O. Box 591, SE-75124 Uppsala, Sweden.
Oliw
Ernst
E
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology; D001233:Aspergillus nidulans / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D049308:Dioxygenases / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D019746:Intramolecular Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019787:Linoleic Acid / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics; D011499:Protein Processing, Post-Translational; D043323:alpha-Mannosidase / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D019787:Linoleic Acid; D010105:Oxygenases; D049308:Dioxygenases; C122014:linoleate diol synthase; D043323:alpha-Mannosidase; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase