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Journal of Bacteriology, March 2001, p. 1961-1973, Vol. 183, No. 6
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.6.1961-1973.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Essential Thioredoxin-Dependent Peroxiredoxin
System from Helicobacter pylori: Genetic and Kinetic
Characterization
Laura M. S.
Baker,1
Ausra
Raudonikiene,2,
Paul S.
Hoffman,2 and
Leslie
B.
Poole1,*
Department of Biochemistry, Wake Forest
University School of Medicine, Winston-Salem, North
Carolina,1 and Department of
Microbiology and Immunology, Dalhousie University, Halifax, Nova
Scotia B3H 4H7, Canada2
Received 18 October 2000/Accepted 3 January 2001
Helicobacter pylori, an oxygen-sensitive
microaerophile, contains an alkyl hydroperoxide reductase homologue
(AhpC, HP1563) that is more closely related to 2-Cys peroxiredoxins of
higher organisms than to most other eubacterial AhpC proteins. Allelic replacement mutagenesis revealed ahpC to be essential,
suggesting a critical role for AhpC in defending H. pylori
against oxygen toxicity. Characterization of the ahpC
promoter region divulged two putative regulatory elements and
identified the transcription initiation site, which was mapped to 96 and 94 bp upstream of the initiation codon. No homologue of
ahpF, which encodes the dedicated AhpC reductase in most
eubacteria, was found in the H. pylori genome. Instead,
homologues of Escherichia coli thioredoxin (Trx) reductase
(TrxR, HP0825) and Trx (Trx1, HP0824) formed a reductase system for
H. pylori AhpC. A second Trx homologue (Trx2, HP1458) was
identified but was incapable of AhpC reduction, although Trx2 exhibited
disulfide reductase activity with other substrates [insulin and
5,5'-dithiobis(2-nitrobenzoic acid)]. AhpC interactions with each
substrate, Trx1 and hydroperoxide, were bimolecular and nonsaturable
(infinite Vmax and Km
values) but rapid enough (at 1 × 105 to 2 × 105 M
1 s
1) to suggest an
important role for AhpC in cellular peroxide metabolism. AhpC also
exhibited a wide specificity for hydroperoxide substrates, which, taken
together with the above results, suggests a minimal binding site for
hydroperoxides composed of little more than the cysteinyl (Cys49)
active site. H. pylori AhpC was not reduced by
Salmonella typhimurium AhpF and was slightly more active
with E. coli TrxR and Trx1 than was S. typhimurium AhpC, demonstrating the specialized catalytic
properties of this peroxiredoxin.
*
Corresponding author. Mailing address: Wake Forest
University School of Medicine, Department of Biochemistry, Medical
Center Blvd., Winston-Salem, NC 27157. Phone: (336) 716-6711. Fax:
(336) 716-7671. E-mail: lbpoole{at}wfubmc.edu.

Present address: Aventis Pasteur Ltd., Toronto, Ontario M2R
3T4,
Canada.
Journal of Bacteriology, March 2001, p. 1961-1973, Vol. 183, No. 6
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.6.1961-1973.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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