Allene oxide synthase from Arabidopsis thaliana (CYP74A1) exhibits dual specificity that is regulated by monomer-micelle association |
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Authors: | Hughes Richard K Belfield Eric J Ashton Ruth Fairhurst Shirley A Göbel Cornelia Stumpe Michael Feussner Ivo Casey Rod |
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Affiliation: | John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom. Richard.Hughes@bbsrc.ac.uk |
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Abstract: | We investigate the effects of detergent on the kinetics and oligomeric state of allene oxide synthase (AOS) from Arabidopsis thaliana (CYP74A1). We show that detergent-free CYP74A1 is monomeric and highly water soluble with dual specificity, but has relatively low activity. Detergent micelles promote a 48-fold increase in k(cat)/K(m) (to 5.9 x 10(7)M(-1)s(-1)) with concomitant changes in the spin state equilibrium of the haem-iron due to the binding of a single detergent micelle to the protein monomer, which is atypical of P450 enzymes. This mechanism is shown to be an important determinant of the substrate specificity of CYP74A1. CYP74A1 may be suited for structural resolution of the first plant cytochrome P450 and its 9-AOS activity and behaviour in vitro has implications for its role in planta. |
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Keywords: | AOS, allene oxide synthase c.m.c., critical micelle concentration CYP74, cytochrome P450 subfamily 74 Emulphogene, polyoxyethylene 10 tridecyl ether HPL, hydroperoxide lyase 13-HPOTE, 13-S-hydroperoxyoctadeca-9Z,11E,15Z-trienoic acid) 13-HPODE, 13-S-hydroperoxyoctadeca-9Z,11E-dienoic acid 9-HPOTE, 9-S-hydroperoxyoctadeca-10E,12Z,15Z-trienoic acid 9-HPODE, 9-S-hydroperoxyoctadeca-10E,12Z-dienoic acid LOX, lipoxygenase RZ, Reinheitszahl or purity index |
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