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Purification and si...
Purification and site-directed mutagenesis of linoleate 9S-dioxygenase-allene oxide synthase of Fusarium oxysporum confirms the oxygenation mechanism
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- Chen, Yang (författare)
- Uppsala universitet,Institutionen för farmaceutisk biovetenskap
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- Jernerén, Fredrik (författare)
- Uppsala universitet,Institutionen för farmaceutisk biovetenskap
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- Oliw, Ernst, 1948- (författare)
- Uppsala universitet,Institutionen för farmaceutisk biovetenskap
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(creator_code:org_t)
- ELSEVIER SCIENCE INC, 2017
- 2017
- Engelska.
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Ingår i: Archives of Biochemistry and Biophysics. - : ELSEVIER SCIENCE INC. - 0003-9861 .- 1096-0384. ; 625-626, s. 24-29
- Relaterad länk:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- 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.
Ämnesord
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
Nyckelord
- Cobalt affinity chromatography
- Cyclooxygenase
- Fusion enzymes
- Jasmonic acid
- Site-directed mutagenesis
- Liquid chromatography-mass spectrometry
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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