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Purification and site-directed mutagenesis of linoleate 9S-dioxygenase-allene oxide synthase of Fusarium oxysporum confirms the oxygenation mechanism
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
2017 (English)In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 625-626, 24-29 p.Article in journal (Refereed) Published
Abstract [en]

Plants and fungi form jasmonic acid from a-linolenic acid. The first two steps of biosynthesis in plants occur by sequential transformation by 13S-Iipoxygenase and allene oxide synthase (AOS). The biosynthesis in fungi may follow this classical scheme, but the only fungal AOS discovered so far are cytochromes P450 (CYP) fused to 8- and 9-dioxygenases (DOX). In the present report, we purified recombinant 9S-DOX-AOS of Fusarium oxysporum from cell lysate by cobalt affinity chromatography to near homogeneity and studied key residues by site-directed mutagenesis. Sequence homology with 8R-DOX-linoleate diol synthases (8R-DOX-LDS) suggested that Tyr414 catalyzes hydrogen abstraction and that Cys1051 forms the heme thiolate ligand. Site-directed mutagenesis (Tyr414Phe; Cys1051Ser) led to loss of 9S-DOX and 9S-AOS activities, respectively, but other important residues in the CYP parts of 5,8 and 7,8-LDS or 9R-AOS were not conserved. The UV-visible spectrum of 9S-DOX-AOS showed a Soret band at 409 nm, which shifted to 413 nm in the Cys1051Ser mutant. The 9S-AOS of the Tyr414Phe mutant transformed 9S-hydroperoxides of alpha-linolenic and linoleic acids to allene oxides/alpha-ketols, but it did not transform 13-hydroperoxides. We conclude that 9S- and 8R-DOX catalyze hydrogen abstraction at C-11 and C-8, respectively, by homologous Tyr residues.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE INC , 2017. Vol. 625-626, 24-29 p.
Keyword [en]
Cobalt affinity chromatography, Cyclooxygenase, Fusion enzymes, Jasmonic acid, Site-directed mutagenesis, Liquid chromatography-mass spectrometry
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-328970DOI: 10.1016/j.abb.2017.05.007ISI: 000404318700004PubMedID: 28502466OAI: oai:DiVA.org:uu-328970DiVA: diva2:1140651
Funder
Swedish Research Council, 03X-06523Knut and Alice Wallenberg Foundation, KAW 2004.123
Available from: 2017-09-12 Created: 2017-09-12 Last updated: 2017-09-12Bibliographically approved

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Jernerén, FredrikOliw, Ernst

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