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Volume 271, Number 46,
Issue of November 15, 1996
pp. 28861-28867
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Xanthophyll Biosynthesis
CLONING, EXPRESSION, FUNCTIONAL RECONSTITUTION, AND REGULATION
OF -CYCLOHEXENYL CAROTENOID EPOXIDASE FROM PEPPER (CAPSICUM
ANNUUM)
(Received for publication, July 8, 1996, and in revised form, August 28, 1996)
Florence
Bouvier
,
Alain
d'Harlingue
§
,
Philippe
Hugueney
¶
,
Elena
Marin
,
Annie
Marion-Poll
and
Bilal
Camara
From the Institut de Biologie Moléculaire des
Plantes du Centre National de la Recherche Scientifique,
Université Louis Pasteur, 12 rue du Général Zimmer,
67084 Strasbourg, France, the § Laboratoire de Pathologie et
Biochimie Végétales, Université Pierre et Marie
Curie, 4 Place Jussieu, 75250 Paris, France, the ¶ Institut
für Biologie II Zellbiologie Schänzlestrasse 1d 79104, Germany, and the Laboratoire de Biologie Cellulaire, Institut
National de la Recherche Agronomique, 78026 Versailles
Cedex, France
Pepper (Capsicum annuum)
-cyclohexenyl xanthophyll epoxidase cDNA was cloned and the
corresponding enzyme overexpressed and purified from Escherichia
coli, for investigation of its catalytic activity. The
recombinant protein did not directly accept NADPH for epoxidation of
cyclohexenyl carotenoids, nor did it operate according to a
peroxygenase-based mechanism. Instead, the reducing power of NADPH was
transferred to the epoxidase via reduced ferredoxin as shown by
reconstitution of epoxidase activity in the presence of NADPH,
ferredoxin oxidoreductase, and ferredoxin. Bacterial rubredoxin could
be substituted for ferredoxin. The pepper epoxidase acted specifically
on the -ring of xanthophylls such as -cryptoxanthin, zeaxanthin,
and antheraxanthin. The proposed reaction mechanism for epoxidation
involves the formation of a transient carbocation. This characteristic
allows selective inhibition of the epoxidase activity by different
nucleophilic diethylamine derivatives,
p-dimethylaminobenzenediazonium fluoroborate and
N,N-dimethyl-2-phenylaziridinium. It was also shown
that the epoxidase gene was up-regulated during oxidative stress and
when chloroplasts undergo differentiation into chromoplasts in pepper
fruit.

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Copyright © 1996 by the American Society for Biochemistry and Molecular Biology.
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