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J. Biol. Chem., Vol. 278, Issue 19, 16658-16666, May 9, 2003
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From the Laboratory of Protein Biochemistry and Protein
Engineering, Ghent University, K. L. Ledeganckstraat 35, 9000 Gent, Belgium
While belonging to the same family of antioxidant
enzymes, members of the peroxiredoxins do not necessarily employ one
and the same method for their reduction. Most representatives become reduced with the aid of thioredoxin, whereas some members use AhpF,
tryparedoxin, or cyclophilin A. Recent research on a new peroxiredoxin
isoform (type C) from Populus trichocarpa has shown that
these particular types may also use glutaredoxin instead of
thioredoxin. This finding is supported by the occurrence of chimeric
proteins composed of a peroxiredoxin and glutaredoxin region. A gene
encoding such a fusion protein is enclosed in the Haemophilus
influenzae Rd genome. We expressed the H. influenzae protein, denoted here as PGdx, in Escherichia coli and
purified the recombinant enzyme. In vitro assays
demonstrate that PGdx, in the presence of dithiothreitol or
glutathione, is able to protect supercoiled DNA against the metal
ion-catalyzed oxidation-system. Enzymatic assays did, indeed,
characterize PGdx as a peroxidase, requiring the glutathione redox
cycle for the reduction of hydrogen peroxide
(kcat/Km 5.01 × 106 s
Purification and Characterization of a Chimeric Enzyme from
Haemophilus influenzae Rd That Exhibits
Glutathione-dependent Peroxidase Activity*
1 M1) as well
as the small organic hydroperoxide tert-butylhydroperoxide (kcat/Km 5.67 × 104 s1 M1).
Enzymatic activity as function of the glutathione concentration deviated from normal Michaelis-Menten kinetics, giving a sigmoidal pattern with an apparent Hill coefficient of 2.9. Besides the formation
of a disulfide-linked PGdx dimer, it was also shown by mass
spectrometric analysis that cysteine 49, which is equivalent to the
active site cysteine of the peroxiredoxins, undergoes glutathionylation during purification under nonreducing conditions. Based on these results, we propose a model for the catalytic mechanism.
*
This work was supported by Institute for the Promotion of
Innovation by Science and Technology in Flanders Grant 3072 (to F. P.)
and by Concerted Research Action 12050198 from Ghent University (to
J. V. B.).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: Laboratory of Protein
Biochemistry and Protein Engineering, Ghent University, K. L. Ledeganckstraat 35, 9000 Gent, Belgium. Tel.: 32-9-264-51-09; Fax:
32-9-264-53-38; E-mail: Jozef.vanbeeumen@rug.ac.be.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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