Original contributionSpecificity and kinetics of a mitochondrial peroxiredoxin of Leishmania infantum
Introduction
The term peroxiredoxin has been coined for a family of homologous proteins that is spread over all living kingdoms [1]. The representative discovered first was the “thiol-specific antioxidant protein” of yeast [2] that was later recognized to be a thioredoxin-dependent peroxidase [3]. The common functional denominator of these proteins appears to be their ability to reduce hydroperoxides at the expenses of thiol substrates [4]. In Kinetoplastida, comprising parasites of the genera Crithidia, Trypanosoma, and Leishmania, the peroxiredoxins so far characterized proved to catalyze the reduction of hydroperoxides by thioredoxin-related proteins called tryparedoxins (TXN) 5, 6, 7, 8, 9, 10, 11. These “tryparedoxin peroxidases” (TXNPx) are the major, if not the only, peroxide detoxifying enzymes in Kinetoplastida. The reduction equivalents are ultimately provided by nicotinamide adenine dinucleotide phosphate (NADPH) as substrate of trypanothione reductase (TR) [12]. Reduced trypanothione [N1,N8-(bis)-glutathionylspermidine] reduces TXN, the substrate of TXNPx [5]. The biological relevance of this unique cascade of oxidoreductases has been corroborated by a conditioned knockout of trypanothione reductase in T. brucei that lead to increased sensitivity to exogenous H2O2 in vitro and elimination of virulence in experimental animals [13].
In C. fasciculata [14], T. cruzi [15], and T. brucei [10] TXN and TXNPx were shown to localize to the cytosol by immunohistochemistry. TR was reported to be predominantly localized in the cytosol of T. brucei [16], but was also found associated with the mitochondrion and the kinetoplast of T. cruzi [17]. The cytosolic localization of the complete system appears ideal to protect the parasite against oxidative attack from outside, as expected from the phagocytic attack by the host. The reduced virulence of TR-deficient T. brucei [13] and of L. donovani with a transdominant mutation in TR [18] may therefore be due in part to an impaired cytosolic peroxide metabolism. Kinetoplastida, like higher animals [19], however, produce endogenous H2O2, also as byproduct of the mitochondrial energy metabolism 20, 21, 22. This endogenous oxidative stress would not be easily balanced by any kind of cytosolic peroxidase. Therefore, it was not surprising that in T. cruzi and T. brucei, in addition to cytosolic enzymes, a mitochondrial peroxiredoxin was found 10, 23. Similarly in L. infantum, a human pathogen prevailing in the Mediterranean countries, two peroxiredoxin genes were identified (Castro et al., [23a]). One of them translates into a cytosolic TXNPx (LicTXNPx; Acc. Nr. AY058210) and is identical to a peroxiredoxin of L. chagasi [24] and almost indistinguishable from those of L. donovani [11] and L. major [8]. The other gene codes for an enzyme (LimTXNPx; Acc. Nr. AY058209) that reminds of the mitochondrial peroxiredoxins of T. cruzi and T. brucei and was indeed shown to localize to this organelle (Castro et al., [23a]). These proteins differ from the typical cytosolic peroxiredoxins in the sequence surrounding their second redox active cysteine and in possessing an N-terminal mitochondrial leader sequence.
Here we report on the heterologous expression, isolation and biochemical characterization of L. infantum mitochondrial peroxiredoxin. The specific questions addressed are: (i) is the peroxiredoxin a TXNPx? (ii) How broad is the specificity range for hydroperoxides? (iii) Is the kinetic pattern compatible with that of other peroxiredoxins? (iv) How does the native enzyme compare with related peroxiredoxins in terms of quaternary structure?
Section snippets
Heterologous expression and purification of LimTXNPx
The complete LimTXNPx coding sequence (Castro et al., [23a]) was amplified with PWO polymerase (GibcoBRL, Paisley, Scotland), using the forward primer and the reverse primer (restriction sites and clamp sequences indicated in lower case; start and stop codons underlined). The PCR product was cloned into the NdeI and XhoI restriction sites of the prokaryotic expression vector pET28a (Novagen, Madison, WI, USA) so that a
LimTXNPx is a tryparedoxin peroxidase
L. infantum presents a 2-Cys mitochondrial peroxiredoxin, LimTXNPx (Castro et al., [23a]), that together with the mitochondrial homologues of T. cruzi and T. brucei 10, 23 forms a peroxiredoxin subfamily, distinct from the cytosolic enzymes from those organisms. Apart from the N-terminal mitochondrial leader sequence, in this subfamily the second redox-active Cys, i.e., the one that interacts with the reductant [4], is not embedded in a Val-Cys-Pro (VCP) motif, as is characteristic of most
Discussion
Peroxiredoxins form a family of peroxidases that act on a variety of hydroperoxides using thiols as cosubstrates, which until now were reported to comprise glutathione, thioredoxin, tryparedoxin, or the CXXC motifs of bacterial AhpF [4]. In this report we refer to a mitochondrial peroxiredoxin of L. infantum (Castro et al., [23a]) that is suggested to form a novel group of TXNPxs together with the homologous molecules of T. cruzi and T. brucei 10, 23. These new peroxiredoxins share with
Abbreviations
AhpC—alkyl hydorperoxide reductase subunit C
AhpF—alkyl hydroperoxide reductase subunit F
BSA—bovine serum albumin
COOH—cumene hydroperoxide
LOOH—linoleic acid hydroperoxide
PCOOH—phosphatidyl choline hydroperoxide
PCR—polymerase chain reaction
t-bOOH—tert-butyl hydroperoxide
TR—trypanothione reductase
TXN—tryparedoxin
TXNPx—tryparedoxin peroxidase
Acknowledgements
We thank R. Brigelius-Flohé for kindly providing phosphatidyl choline hydroperoxide and linoleic acid hydroperoxide. We acknowledge financial support from Deutsche Forschungsgemeinschaft (Grant FL 61/8-3) and from Fundação para a Ciĉncia e a Tecnologia (FCT) (Grant PRAXIS/P/SAU/10263/1998), Portugal. H. Castro is recipient of a FCT doctoral fellowship (Grant SFRH/BD/1396/2000).
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