The major C18 cutin monomers are 18-hydroxy-9,10-epoxystearic and 9,10,18-trihydroxystearic acids. These compounds are also known messengers in plant-pathogen interactions. We have previously shown that their common precursor 9,10-epoxystearic acid was formed by the epoxidation of oleic acid in Vicia sativa microsomes (Pinot, Salaün, Bosch, Lesot, Mioskowski and Durst (1992) Biochem. Biophys. Res. Commun. 184, 183-193). Here we determine the chirality of the epoxide produced as (9R,10S) and (9S,10R) in the ratio 90:10 respectively. We further show that microsomes from yeast expressing the cytochrome P450 CYP94A1 are capable of hydroxylating the methyl terminus of 9,10-epoxystearic and 9,10-dihydroxystearic acids in the presence of NADPH to form the corresponding 18-hydroxy derivatives. The reactions were not catalysed by microsomes from yeast transformed with a void plasmid or in absence of NADPH. After incubation of a synthetic racemic mixture of 9,10-epoxystearic acid with microsomes of yeast expressing CYP94A1, the chirality of the residual epoxide was shifted to 66:34 in favour of the (9S,10R) enantiomer. Both enantiomers were incubated separately and Vmax/Km values of 16 and 3.42 ml/min per nmol of P450 for (9R,10S) and (9S,10R) respectively were determined, demonstrating that CYP94A1 is enantioselective for the (9R,10S) enantiomer, which is preferentially formed in V. sativa microsomes. Compared with the epoxide, the diol 9,10-dihydroxystearic acid was a much poorer substrate for the ω-hydroxylase, with a measured Vmax/Km of 0.33 ml/min per nmol of P450. Our results indicate that the activity of CYP94A1 is strongly influenced by the stereochemistry of the 9,10-epoxide and the nature of substituents on carbons 9 and 10, with Vmax/Km values for epoxide ≫ oleic acid > diol.
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August 1999
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Research Article|
August 10 1999
Production in vitro by the cytochrome P450 CYP94A1 of major C18 cutin monomers and potential messengers in plant–pathogen interactions: enantioselectivity studies
Franck PINOT;
Franck PINOT
1
*Institut de Biologie Moléculaire des Plantes-CNRS UPR406, Département d'Enzymologie Cellulaire et Moléculaire, 28 rue Goethe, F-67083 Strasbourg Cedex, France
1To whom correspondence should be addressed (e-mail franck.pinot@bota-ulp.u-strasbg.fr).
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Irène BENVENISTE;
Irène BENVENISTE
*Institut de Biologie Moléculaire des Plantes-CNRS UPR406, Département d'Enzymologie Cellulaire et Moléculaire, 28 rue Goethe, F-67083 Strasbourg Cedex, France
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Jean-Pierre SALAüN;
Jean-Pierre SALAüN
*Institut de Biologie Moléculaire des Plantes-CNRS UPR406, Département d'Enzymologie Cellulaire et Moléculaire, 28 rue Goethe, F-67083 Strasbourg Cedex, France
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Olivier LOREAU;
Olivier LOREAU
†CEA Saclay, Service des molécules marquées, Bâtiment 547, 91191 Gif sur Yvette Cedex, France
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Jean-Pierre NOËL;
Jean-Pierre NOËL
†CEA Saclay, Service des molécules marquées, Bâtiment 547, 91191 Gif sur Yvette Cedex, France
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Lukas SCHREIBER;
Lukas SCHREIBER
‡Julius-von-Sachs-Institut für Biowissenschaften, Lehrstuhl für Botanik II, Universität Würzburg, Julius-von-Sachs-Platz 3, D-97082 Würzburg, Germany
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Francis DURST
Francis DURST
*Institut de Biologie Moléculaire des Plantes-CNRS UPR406, Département d'Enzymologie Cellulaire et Moléculaire, 28 rue Goethe, F-67083 Strasbourg Cedex, France
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Publisher: Portland Press Ltd
Received:
March 17 1999
Revision Received:
June 01 1999
Accepted:
June 14 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 342 (1): 27–32.
Article history
Received:
March 17 1999
Revision Received:
June 01 1999
Accepted:
June 14 1999
Citation
Franck PINOT, Irène BENVENISTE, Jean-Pierre SALAüN, Olivier LOREAU, Jean-Pierre NOËL, Lukas SCHREIBER, Francis DURST; Production in vitro by the cytochrome P450 CYP94A1 of major C18 cutin monomers and potential messengers in plant–pathogen interactions: enantioselectivity studies. Biochem J 15 August 1999; 342 (1): 27–32. doi: https://doi.org/10.1042/bj3420027
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