Pichia expression of and mutagenesis of 7,8-linoleate diol synthase change the dioxygenase and hydroperoxide isomerase
2008 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 373, no 4, 579-583 p.Article in journal (Refereed) Published
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 α-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.
Place, publisher, year, edition, pages
2008. Vol. 373, no 4, 579-583 p.
Aspergillus nidulans, Dioxygenase, Gaeumannomyces graminis, Hemoprotein, Hydroperoxide isomerase, HPLC, α-Mannosidase, Mass-spectrometry, Myeloperoxidase
IdentifiersURN: urn:nbn:se:uu:diva-86644DOI: 10.1016/j.bbrc.2008.06.060ISI: 000258208500023PubMedID: 18586008OAI: oai:DiVA.org:uu-86644DiVA: diva2:126743