Original contribution
Specificity and kinetics of a mitochondrial peroxiredoxin of Leishmania infantum

https://doi.org/10.1016/S0891-5849(02)01088-2Get rights and content

Abstract

In Kinetoplastida, comprising the medically important parasites Trypanosoma brucei, T. cruzi, and Leishmania species, 2-Cys peroxiredoxins described to date have been shown to catalyze reduction of peroxides by the specific thiol trypanothione using tryparedoxin, a thioredoxin-related protein, as an immediate electron donor. Here we show that a mitochondrial peroxiredoxin from L. infantum (LimTXNPx) is also a tryparedoxin peroxidase. In an heterologous system constituted by nicotinamide adenine dinucleotide phosphate (NADPH), T. cruzi trypanothione reductase, trypanothione and Crithidia fasciculata tryparedoxin (CfTXN1 and CfTXN2), the recombinant enzyme purified from Escherichia coli as an N-terminally His-tagged protein preferentially reduces H2O2 and tert-butyl hydroperoxide and less actively cumene hydroperoxide. Linoleic acid hydroperoxide and phosphatidyl choline hydroperoxide are poor substrates in the sense that they are reduced weakly and inhibit the enzyme in a concentration- and time-dependent way. Kinetic parameters deduced for LimTXNPx are a kcat of 37.0 s−1 and Km values of 31.9 and 9.1 μM for CfTXN2 and tert-butyl hydroperoxide, respectively. Kinetic analysis indicates that LimTXNPx does not follow the classic ping-pong mechanism described for other TXNPx (Φ1,2 = 0.8 s·μM2). Although the molecular mechanism underlying this finding is unknown, we propose that cooperativity between the redox centers of subunits may explain the unusual kinetic behavior observed. This hypothesis is corroborated by high-resolution electron microscopy and gel chromatography that reveal the native enzyme to preferentially exist as a homodecameric ring structure composed of five dimers.

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 5′-ccgcgcacat ATGCTCCGCCGTCTTCCCAOH and the reverse primer 5′-caccgctcgagTCACATGTTCTTCTCGAAAAACOH (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).

References (46)

  • A Denicola-Seoane et al.

    Succinate-dependent metabolism in Trypanosoma cruzi epimastigotes

    Mol. Biochem. Parasitol.

    (1992)
  • S.R Wilkinson et al.

    Distinct mitochondrial and cytosolic enzymes mediate trypanothione-dependent peroxide metabolism in Trypanosoma cruzi

    J. Biol. Chem.

    (2000)
  • H Castro et al.

    Complementary antioxidant defense by cytoplasmic and mitochondrial peroxiredoxins in Leishmania infantum

    Free Radic. Biol. Med.

    (2002)
  • S.D Barr et al.

    Cloning and characterization of three differentially expressed peroxidoxin genes from Leishmania chagasievidence for an enzymatic detoxification of hydroxyl radicals

    J. Biol. Chem.

    (2001)
  • F Ursini et al.

    The selenoenzyme phospholipid hydroperoxide glutathione peroxidase

    Biochim. Biophys. Acta

    (1985)
  • A Holmgren et al.

    Thioredoxin and thioredoxin reductase

    Methods Enzymol

    (1995)
  • M Montemartini et al.

    Sequence analysis of the tryparedoxin peroxidase gene from Crithidia fasciculata and its functional expression in Escherichia coli

    J. Biol. Chem.

    (1998)
  • M.S Alphey et al.

    The structure of reduced tryparedoxin peroxidase reveals a decamer and insight into reactivity of 2 Cys-peroxiredoxins

    J. Mol. Biol.

    (2000)
  • J.R Harris et al.

    Comparison of the decameric structure of peroxiredoxin-II by transmission electron microscopy and X-ray crystallography

    Biochim. Biophys. Acta

    (2001)
  • H.Z Chae et al.

    Cloning and sequencing of thiol-specific antioxidant enzymes

    Proc. Natl. Acad. Sci. USA

    (1994)
  • B Hofmann et al.

    Peroxiredoxins

    Biol. Chem.

    (2002)
  • E Nogoceke et al.

    A unique cascade of oxireductases catalyses trypanothione-mediated peroxide metabolism in Crithidia fasciculta

    Biol. Chem.

    (1997)
  • H Ldemann et al.

    Trypanosoma brucei tryparedoxin, a thioredoxin-like protein in African trypanosomes

    FEBS Lett

    (1998)
  • Cited by (55)

    • Thiol oxidation by biologically-relevant reactive species

      2022, Redox Chemistry and Biology of Thiols
    • The mitochondrial peroxiredoxin displays distinct roles in different developmental stages of African trypanosomes

      2020, Redox Biology
      Citation Excerpt :

      Hydroperoxides are detoxified by Prxs [9,10] and non-selenium glutathione peroxidase-type (Px) enzymes [10–12]. Whereas the latter enzymes preferably detoxify lipid-derived hydroperoxides [13–15], the Prxs use hydrogen peroxide and peroxynitrite as main substrates [16,17]. Both types of peroxidases obtain their reducing equivalents from the T(SH)2/Tpx couple and thus act as tryparedoxin peroxidases [6,8,11,18,19].

    • Oligomerization dynamics and functionality of Trypanosoma cruzi cytosolic tryparedoxin peroxidase as peroxidase and molecular chaperone

      2019, Biochimica et Biophysica Acta - General Subjects
      Citation Excerpt :

      It should be noted that even during incubation with increasing amount of DTT (up to 100 mM) and SDS (up to 4%), we still observed oligomers of the enzyme, an indication that there are strong interactions between subunits which are not disrupted by reducing conditions (data not shown). The presence of oligomeric products in SDS-PAGE, even under reducing conditions, was also observed for other kinetoplastid 2-Cys Prx proteins [11,32–35]. It may therefore be inferred that this is a particular feature of kinetoplastid Prxs.

    • Immunization with the Leishmania infantum recombinant cyclophilin protein 1 confers partial protection to subsequent parasite infection and generates specific memory T cells

      2014, Vaccine
      Citation Excerpt :

      Upon transformation of Escherichia coli BL21, a modified recombinant form LiCyP1 containing an amino-terminal six-histidine tag was produced. The protein used throughout this work was purified from bacteria cultures containing 50 μg ml−1 kanamycin 0.1% glucose and induced overnight at 18 °C with 0.2 mM isopropyl-B-d-thiogalactopyranoside [3]. Briefly, bacteria was pelleted, suspended in 500 mM NaCl, 20 mM Tris–HCl, disrupted by sonication and centrifuged at 10,000 × g 30 min at 4 °C.

    View all citing articles on Scopus
    View full text