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J. Biol. Chem., Vol. 275, Issue 37, 28715-28721, September 15, 2000
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From the The citrus phospholipid hydroperoxide glutathione
peroxidase (cit-PHGPx) was the first plant peroxidase demonstrated to
exhibit PHGPx-specific enzymatic activity, although it was 500-fold
weaker than that of the pig heart analog. This relatively low activity is accounted for the catalytic residue of cit-PHGPx, which was found to
be cysteine and not the rare selenocysteine (Sec) present in animal
enzymes. Sec incorporation into proteins is encoded by a UGA codon,
usually a STOP codon, which, in prokaryotes, is suppressed by an
adjacent downstream mRNA stem-loop structure, the Sec insertion
sequence (SECIS). By performing appropriate nucleotide substitutions
into the gene encoding cit-PHGPx, we introduced bacterial-type SECIS
elements that afforded the substitution of the catalytic
Cys41 by Sec, as established by mass spectrometry,
while preserving the functional integrity of the peroxidase. The
recombinant enzyme, whose synthesis is selenium-dependent,
displayed a 4-fold enhanced peroxidase activity as compared with the
Cys-containing analog, thus confirming the higher catalytic power of
Sec compared with Cys in cit-PHGPx active site. The study led also to
refinement of the minimal sequence requirements of the bacterial-type
SECIS, and, for the first time, to the heterologous expression in
Escherichia coli of a eukaryotic selenoprotein containing a
SECIS in its open reading frame.
Substituting Selenocysteine for Catalytic Cysteine 41 Enhances
Enzymatic Activity of Plant Phospholipid Hydroperoxide Glutathione
Peroxidase Expressed in Escherichia coli*
§,,
Department of Fruit Tree Breeding and
Molecular Genetics, Agricultural Research Organization, The
Volcani Center, 50250 Bet-Dagan, Israel and the ¶ Institut Cochin
de Genetique Moleculaire, CNRS UPR 415, 22 rue Mechain,
75014 Paris, France
*
This work was supported by the Chief Scientist of the
Israeli Ministry of Agriculture Biotechnology Fund.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Fruit
Tree Breeding and Molecular Genetics, A.R.O., P.O. Box 6, The Volcani Center, 50250 Bet-Dagan, Israel. Tel.: 972-3-968-3777; Fax:
972-3-966-9583; E-mail: vhyuval@agri.gov.il.
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