Sequence Analysis of the Tryparedoxin Peroxidase Gene fromCrithidia fasciculata and Its Functional Expression in Escherichia coli

Montemartini, Marisa; Nogoceke, Everson; Singh, Mahavir; Steinert, Peter M.; Flohé, Leopold; Kalisz, Henryk M.

doi:10.1074/jbc.273.9.4864

Cited by 63 publications

(48 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Despite the use of His-tagged proteins, the Prx activities are assumed to be similar to those of native Prxs as shown in the comparative work with Prxs of Crithidia fasciculata by Montemartini et al (1998), in which N-terminally His-tagged Prx was shown to be as active as the purified Prx protein. Nevertheless, the use of His-tagged proteins should be kept in mind and the enzymic data interpreted with caution.…”

Section: The Catalytic Activity Of Prxmentioning

confidence: 99%

Divergent Light-, Ascorbate-, and Oxidative Stress-Dependent Regulation of Expression of the Peroxiredoxin Gene Family in Arabidopsis

et al. 2003

View full text Add to dashboard Cite

Peroxiredoxins (prxs) are peroxidases with broad substrate specificity. The seven prx genes expressed in Arabidopsis shoots were analyzed for their expressional response to changing photon fluence rates, oxidative stress, and ascorbate application. The results reveal a highly variable and gene-specific response to reducing and oxidizing conditions. The steady-state transcript amounts of the chloroplast-targeted prxs, namely the two-cysteine (2-Cys) prxs, prx Q and prx II E, decreased upon application of ascorbate. prx Q also responded to peroxides and diamide treatment. prx II B was induced by tertiary butylhydroperoxide, but rather unaffected by ascorbate. The strongest responses were observed for prx II C, which was induced with all treatments. The two Arabidopsis 2-Cys Prxs and four Prx II proteins were expressed heterologously in Escherichia coli. In an in vitro test system, they all showed peroxidase activity, but could be distinguished by their ability to accept dithiothreitol and thioredoxin as electron donor in the regeneration reaction. The midpoint redox potentials (E m Ј) of Prx II B, Prx II C, and Prx II E were around Ϫ290 mV and, thus, less negative than E m Ј of Prx II F, 2-Cys Prx A, and 2-Cys Prx B (Ϫ307 to Ϫ322 mV). The data characterize expression and function of the mitochondrial Prx II F and the chloroplast Prx II E for the first time, to our knowledge. Antibodies directed against 2-Cys Prx and Prx II C showed a slight up-regulation of Prx II protein in strong light and of 2-Cys Prx upon transfer both to high and low light. The results are discussed in context with the subcellular localization of the Prx gene products.Peroxiredoxins (Prxs) are enzymes that reduce hydrogen peroxide (H 2 O 2 ) and alkyl hydroperoxides. They are grouped in four classes: (a) 2-Cys Prx; (b) Prx Q; (c) Prx II, which all contain two catalytic Cys residues in distinct sequence environment; and (d) 1-Cys Prx with one conserved Cys residue only (Dietz, 2003). A phylogenetic distance analysis suggests that 2-Cys Prx, Prx Q, and 1-Cys Prx are related proteins, whereas the group of Prx II is likely to have evolved independently (Verdoucq et al., 1999;Horling et al., 2002). The catalytic Cys residues undergo oxidation during the peroxide reduction reaction and need to be reduced by electron donors such as glutaredoxins, thioredoxins, or cyclophilins before the next catalytic cycle (Lee et al., 2001;Rouhier et al., 2001;Kö nig et al., 2002). For the bacterial and animal homologs, a broad substrate specificity has been described (Nogoceke et al., 1997; Bryk et al., 2000; Hillas et al., 2000). In in vitro tests, these Prx proteins reduced H 2 O 2 , lipid peroxides, such as butyl hydroperoxide, phospholipid peroxides and cumene hydroperoxide, and peroxynitrite. For plant Prxs, the catalytic properties have only poorly been investigated.The Arabidopsis genome encodes 10 open reading frames (ORFs) for peroxiredoxins. Based on sequence similarities, they can be assigned to the four subgroups of peroxiredoxins: two ORFs code for 2...

show abstract

Section: The Catalytic Activity Of Prxmentioning

confidence: 99%

Divergent Light-, Ascorbate-, and Oxidative Stress-Dependent Regulation of Expression of the Peroxiredoxin Gene Family in Arabidopsis

et al. 2003

View full text Add to dashboard Cite

show abstract

“…The medically important trypanosomatids do not contain catalase, glutathione peroxidase, or glutathione reductase but rely on an analogous system to regulate oxidative stress. The details of this trypanothione peroxidase system have been elucidated in the model trypanosome Crithidia fasciculata (6,8,10,11) and are shown in Fig. 1.…”

mentioning

confidence: 99%

The High Resolution Crystal Structure of Recombinant Crithidia fasciculata Tryparedoxin-I

Alphey¹,

Leonard²,

Gourley³

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

Tryparedoxin-I is a recently discovered thiol-disulfide oxidoreductase involved in the regulation of oxidative stress in parasitic trypanosomatids. The crystal structure of recombinant Crithidia fasciculata tryparedoxin-I in the oxidized state has been determined using multi-wavelength anomalous dispersion methods applied to a selenomethionyl derivative. The model comprises residues 3 to 145 with 236 water molecules and has been refined using all data between a 19-and 1.4-Å resolution to an R-factor and R-free of 19.1 and 22.3%, respectively. Despite sharing only about 20% sequence identity, tryparedoxin-I presents a five-stranded twisted ␤-sheet and two elements of helical structure in the same type of fold as displayed by thioredoxin, the archetypal thiol-disulfide oxidoreductase. However, the relationship of secondary structure with the linear amino acid sequences is different for each protein, producing a distinctive topology. The ␤-sheet core is extended in the trypanosomatid protein with an N-terminal ␤-hairpin. There are also differences in the content and orientation of helical elements of secondary structure positioned at the surface of the proteins, which leads to different shapes and charge distributions between human thioredoxin and tryparedoxin-I. A righthanded redox-active disulfide is formed between Cys-40 and Cys-43 at the N-terminal region of a distorted ␣-helix (␣1). Cys-40 is solvent-accessible, and Cys-43 is positioned in a hydrophilic cavity. Three C-H⅐⅐⅐O hydrogen bonds donated from two proline residues serve to stabilize the disulfide-carrying helix and support the correct alignment of active site residues. The accurate model for tryparedoxin-I allows for comparisons with the family of thiol-disulfide oxidoreductases and provides a template for the discovery or design of selective inhibitors of hydroperoxide metabolism in trypanosomes. Such inhibitors are sought as potential therapies against a range of human pathogens.Parasitic trypanosomatids, belonging to the order Kinetoplastida, cause debilitating and life-threatening human diseases such as African sleeping sickness, Chagas' disease, and the leishmaniases (1). The current therapies against these infections are inadequate due to poor drug efficacy and toxicity combined with increasing drug resistance (2). There is therefore an urgent need to understand how drugs already in use function so that they might be improved and to identify new targets for chemotherapeutic attack. The ideal target is an enzyme of a metabolic pathway that is essential for the survival of the parasite and either absent in the human host or one that presents differing substrate specificities (3). Because trypanosomatids are susceptible to oxidative stress, this aspect of their metabolism represents an attractive target for the development of new trypanocidal agents (4 -6).As with other organisms living in an aerobic environment, trypanosomes are exposed to reactive oxygen intermediates such as superoxide anion and hydrogen peroxide. These potentially destructive c...

show abstract

“…All three TXNs have been shown to be specifically reduced by trypanothione with comparably low K m values. TXN1 and TXN2 can substitute for each other in the reduction of tryparedoxin peroxidase [11], whereas the TXN of T. brucei was reported to act as a reducing substrate for the ribonucleotide reductase [9]. It appears conceivable that the diversification of the TXNs provides the basis for functional specialization allowing specific reduction of distinct disulfides at the expense of trypanothione.…”

Section: Discussionmentioning

confidence: 99%

“…The correspondence of the deduced amino acid sequence with the peptides derived from authentic TXN1 leaves no doubt that this time we have indeed cloned the TXN1 gene. The single amino acid exchange detected at position 142 is either explained by the ambiguities resulting from sequencing traces of peptides or due to common microheterogeneities in variable regions of kinetoplast proteins encoded by multicopy genes [11,14]. The availability of three distinct TXN sequences now allows a better classification of these novel proteins.…”

Section: Discussionmentioning

confidence: 99%

“…Partial DNA sequencing of the TXN1 gene Genomic DNA of C. fasciculata was isolated as described previously by Montemartini et al [11] and used as a template for PCR. Based on the partial peptide sequences of TXN1 [7] degenerate and specific PCR primers were designed (forward: 5 H -GGSAARCTRGTSTTCTTCTACTTC-3 H and reverse: 5 H -C-TCGCTCTGCGCAAACGG-3 H ) to yield a PCR product corresponding to 50% of the TXN1 reading frame.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Sequence, heterologous expression and functional characterization of tryparedoxin1 from Crithidia fasciculata

Guerrero¹,

Flohé²,

Kalisz³

et al. 1999

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

Tryparedoxin (TXN) has recently been discovered as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke et al. (1997) Biol. Chem. 378, 827±836] where it catalyzes the reduction of a peroxiredoxin-type peroxidase by trypanothione. Here we report on the full-length DNA sequence of the TXN previously isolated from C. fasciculata (TXN1). The deduced amino acid sequence comprises 147 residues and matches with all the peptide sequences of fragments obtained from TXN1. It shares a characteristic sequence motif YFSAxWCPPCR with some thioredoxin-related proteins of unknown function. This motif is homologous with the CXXC motif, which characterizes the thioredoxin superfamily of proteins and is known to catalyze disulfide reductions. Sequence conservations between TXNs and the typical thioredoxins are restricted to the intimate environment of the CXXC motif and three more remote residues presumed to contribute to the folding pattern of the thioredoxin-type proteins. The TXNs thus form a distinct molecular clade within the thioredoxin superfamily. TXN1 was expressed in Escherichia coli BL21(DE3)pLysS as a C-terminally extended and His-tagged protein, isolated by chelate chromatography and characterized functionally. The recombinant product exhibited a kinetic pattern identical with, and kinetic parameters similar to those of the authentic enzyme in the trypanothione/peroxiredoxin oxidoreductase assay. The recombinant TXN1 can therefore be considered a valuable tool for the screening of specific inhibitors as potential trypanocidal agents.

show abstract

Sequence Analysis of the Tryparedoxin Peroxidase Gene fromCrithidia fasciculata and Its Functional Expression in Escherichia coli

Abstract: Tryparedoxin peroxidase from

Cited by 63 publications

References 27 publications

Divergent Light-, Ascorbate-, and Oxidative Stress-Dependent Regulation of Expression of the Peroxiredoxin Gene Family in Arabidopsis

Divergent Light-, Ascorbate-, and Oxidative Stress-Dependent Regulation of Expression of the Peroxiredoxin Gene Family in Arabidopsis

The High Resolution Crystal Structure of Recombinant Crithidia fasciculata Tryparedoxin-I

Sequence, heterologous expression and functional characterization of tryparedoxin1 from Crithidia fasciculata

Contact Info

Product

Resources

About