Overproduction, purification, crystallization and preliminary X-ray analysis of the peroxiredoxin domain of a larger natural hybrid protein from Thermotoga maritima.
ABSTRACT Thermotoga maritima contains a natural hybrid protein constituted of two moieties: a peroxiredoxin domain at the N-terminus and a nitroreductase domain at the C-terminus. The peroxiredoxin (Prx) domain has been overproduced and purified from Escherichia coli cells. The recombinant Prx domain, which is homologous to bacterial Prx BCP and plant Prx Q, folds properly into a stable protein that possesses biological activity. The recombinant protein was crystallized and synchrotron data were collected to 2.9 A resolution. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 176.67, c = 141.20 A.
- SourceAvailable from: Jean-Pierre Jacquot[show abstract] [hide abstract]
ABSTRACT: Thiol peroxidases are ubiquitous recently characterized heme-free peroxidases, which catalyze the reduction of peroxynitrites and of various peroxides by catalytic cysteine residues and thiol-containing proteins as reductants. In plants, five different classes can be distinguished, according to the number and the position of conserved catalytic cysteines. Four classes are defined as peroxiredoxins and were already identified by phylogenetic sequence analysis, 1-Cys, 2-Cys, type II, and type Q peroxiredoxins, and the fifth is represented by glutathione peroxidases, which were recently shown to possess a thioredoxin-dependent activity in plants. Since the discovery of peroxiredoxins in plants in 1996, a lot of work has been devoted to the biochemical and functional characterization of the different peroxiredoxin isoforms, but in contrast, few structural data are available. The analysis of the Arabidopsis thaliana genome indicates that at least 17 isoforms of thioredoxin-dependent peroxidases are expressed in various plant compartments. The role of these proteins is discussed in terms of electron donor and substrate specificities and in light of their expression and localization. These enzymes are expressed in many plant tissues and are involved notably in the protection of the photosynthetic apparatus, in the response to various biotic or abiotic stresses by fighting reactive oxygen or nitrogen species and lipid peroxidation.Free Radical Biology and Medicine 07/2005; 38(11):1413-21. · 5.27 Impact Factor
Article: Peroxiredoxins.[show abstract] [hide abstract]
ABSTRACT: Present knowledge on peroxiredoxins is reviewed with special emphasis on catalytic principles, specificities and biological function. Peroxiredoxins are low efficiency peroxidases using thiols as reductants. They appear to be fairly promiscuous with respect to the hydroperoxide substrate; the specificities for the donor substrate vary considerably between the subfamilies, comprising GSH, thioredoxin, tryparedoxin and the analogous CXXC motifs in bacterial AhpF proteins. Peroxiredoxins are definitely responsible for antioxidant defense in bacteria (AhpC), yeast (thioredoxin peroxidase) and trypanosomatids (tryparedoxin peroxidase). They are considered to determine virulence of mycobacteria and trypanosomatids. In higher plants they are involved in balancing hydroperoxide production during photosynthesis. In higher animals peroxiredoxins appear to be involved in the redox-regulation of cellular signaling and differentiation, displaying in part opposite effects.Biological Chemistry 01/2002; 383(3-4):347-64. · 2.96 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Escherichia coli bacterioferritin comigratory protein (BCP), a putative bacterial member of the TSA/AhpC family, was characterized as a thiol peroxidase. BCP showed a thioredoxin-dependent thiol peroxidase activity. BCP preferentially reduced linoleic acid hydroperoxide rather than H(2)O(2) and t-butyl hydroperoxide with the use of thioredoxin as an in vivo immediate electron donor. The value of V(max)/K(m) of BCP for linoleic acid hydroperoxide was calculated to be 5-fold higher than that for H(2)O(2), implying that BCP has a selective capability to reduce linoleic acid hydroperoxide. Replacement of Cys-45 with serine resulted in the complete loss of thiol peroxidase activity, suggesting that BCP is a new bacterial member of TSA/AhpC family having a conserved cysteine as the primary site of catalysis. BCP exists as a monomer, and its functional Cys-45 appeared to exist as cysteine sulfenic acid. The expression level of BCP gradually elevated during exponential growth until mid-log phase growth, beyond which the expression level was decreased. BCP was induced 3-fold by the oxidative stress given by changing the growth conditions from the anaerobic to aerobic culture. Bcp null mutant grew more slowly than its wild type in aerobic culture and showed the hypersensitivity toward various oxidants such as H(2)O(2), t-butyl hydroperoxide, and linoleic acid hydroperoxide. The peroxide hypersensitivity of the null mutant could be complemented by the expression of bcp gene. Taken together, these data suggest that BCP is a new member of thioredoxin-dependent TSA/AhpC family, acting as a general hydroperoxide peroxidase.Journal of Biological Chemistry 02/2000; 275(4):2924-30. · 4.65 Impact Factor
Acta Crystallographica Section F
Editors: H. M. Einspahr and J. M. Guss
Overproduction, purification, crystallization and preliminary
X-ray analysis of the peroxiredoxin domain of a larger natural
hybrid protein from Thermotoga maritima
Carole Barbey, Nicolas Rouhier, Ahmed Haouz, Alda Navaza and
Acta Cryst. (2008). F64, 29–31
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Acta Cryst. (2008). F64, 29–31Barbey et al. · Peroxiredoxin domain of natural hybrid protein
Acta Cryst. (2008). F64, 29–31doi:10.1107/S1744309107064391
Acta Crystallographica Section F
Overproduction, purification, crystallization and
preliminary X-ray analysis of the peroxiredoxin
domain of a larger natural hybrid protein from
Carole Barbey,a* Nicolas
Alda Navazaaand Jean-Pierre
aLaboratoire de Biophysique Mole ´culaire,
Cellulaire et Tissulaire, UMR 7033, Universite ´
Paris 13, UFR SMBH, 74 Rue Marcel Cachin,
93017 Bobigny CEDEX, France,bUnite ´ Mixte de
Recherches 1136 INRA UHP (Interaction Arbres
Microorganismes), IFR 110, Nancy Universite ´
BP 239, 54506 Vandoeuvre-le `s-Nancy CEDEX,
France, andcPlate-forme de Cristallogene `se et
Diffraction des Rayons X, Institut Pasteur,
25 Rue du Dr Roux, 75724 Paris, France
Received 21 September 2007
Accepted 29 November 2007
Thermotoga maritima contains a natural hybrid protein constituted of two
moieties: a peroxiredoxin domain at the N-terminus and a nitroreductase
domain at the C-terminus. The peroxiredoxin (Prx) domain has been
overproduced and purified from Escherichia coli cells. The recombinant Prx
domain, which is homologous to bacterial Prx BCP and plant Prx Q, folds
properly into a stable protein that possesses biological activity. The recombinant
protein was crystallized and synchrotron data were collected to 2.9 A˚resolution.
The crystals belonged to the tetragonal space group I422, with unit-cell
parameters a = b = 176.67, c = 141.20 A˚.
Peroxiredoxins (Prxs) are non-haem-containing peroxidases that
generally catalyze the reduction of peroxides to the corresponding
alcohols through a catalytic cysteine that becomes oxidized to a
sulfenic acid during the catalytic process (Chae et al., 1994; Hofmann
et al., 2002; Wood et al., 2003). Depending on their subunit compo-
sition and the mode of regeneration of the catalytic cysteine,
peroxiredoxins have been classified into three types. The first group
comprises the so-called typical 2-Cys peroxiredoxins, which are
dimeric enzymes with a disulfide bridging two identical subunits. The
second group, called 1-Cys peroxiredoxins, includes Prxs in which
only one catalytic cysteine is conserved. The regenerating system
used by the 1-Cys Prxs remains unknown, although ascorbate and
glutathione have been shown to be efficient in some cases (Kang et
al., 1998; Monteiro et al., 2007). The last group, called atypical 2-Cys
peroxiredoxins, mostly contains Prxs that form an intramolecular
disulfide bridge between two conserved cysteines. Nevertheless, in
the case of plant type II Prxs, which were initially thought to belong
to this group, the catalytic cysteine is conserved and essential but
mutation of the noncatalytic cysteine does not totally abolish the
activity (Rouhier et al., 2002). This led to the introduction of another
group of Prxs in plants, the Prx type II subgroup, which are Prxs that
use only one cysteine (Rouhier & Jacquot, 2002). Usually, the
thioredoxin system is needed for the reduction of the disulfide
formed during catalysis and the regeneration of a reduced ‘active’
enzyme, but poplar type II Prx can use either thioredoxin or gluta-
redoxin as reductants (Rouhier et al., 2001). The atypical 2-Cys Prx
subgroup also contains the so-called glutathione peroxidases from
some organisms such as plants, yeast and probably bacteria, which are
in fact thioredoxin-dependent peroxidases and constitute another
subgroup of the peroxiredoxin or thiol-peroxidase family (Rouhier &
Jacquot, 2005; Herbette et al., 2002; Navrot et al., 2006; Tanaka et al.,
Finally, the atypical 2-Cys Prxs also include a different type of Prx,
known as Prx BCP (bacterioferritin comigratory proteins) in bacteria
or Prx Q in plants. These have been described as monomeric enzymes
and contain one or two conserved cysteines depending on the species.
The first is the catalytic cysteine and the second, when present, is
located five amino acids downstream (in Escherichia coli and in
poplar). In the poplar enzyme the mutation of the first of these
cysteines results in an inactive enzyme, but the enzyme lacking the
second cysteine is still active provided the thioredoxin concentration
# 2008 International Union of Crystallography
All rights reserved
is increased (Rouhier et al., 2004). Interestingly, the second cysteine
mutant can also be reduced via glutaredoxin, while the wild-type
protein is only regenerated via the thioredoxin system. Nevertheless,
an intramolecular disulfide bridge can be formed between these two
cysteines in plant enzymes and, as the presence of these two cysteines
leads to a more active enzyme, the physiological regeneration
mechanism used by the enzyme would preferably involve the
formation of this particular disulfide bridge. In E. coli, it has been
shown that a mutated protein possessing only the peroxidatic cysteine
is still active and is regenerated by thioredoxin (Jeong et al., 2000).
Again, the classification is not adapted to this class of Prx as no
intramolecular disulfide bridge can be formed in proteins that possess
only one conserved cysteine. These intriguing biochemical data raise
interest concerning the function of Prx BCP sequences in which the
second cysteine is naturally absent. One of these sequences is present
in Thermotoga maritima as a domain of a larger protein (321 amino
acids; Genbank accession No. AAD35471) constituted of a natural
hybrid between a Prx BCP module at the N-terminus (?140 amino
acids) and a nitroreductase module at the C-terminus (?170 amino
acids). Here, we present crystallization and diffraction data
concerning the Prx BCP module of this larger protein.
2. Experimental procedures
2.1. Cloning, overproduction and purification
The Prx module of a hybrid protein from T. maritima constituted of
a peroxiredoxin domain followed by a nitroreductase domain was
cloned separately in pET3d using the primers 50-CCCCCCATGGC-
CTTCTTCTATCAGCG-30(NcoI and BamHI restriction sites shown
in bold). This module starts with the amino-acid sequence
MARVKHF and ends with RLIEED. As many arginine codons
unfavourable for expression in E. coli (AGG and AGA) are present
in this sequence, the protein was overproduced in the E. coli
BL21(DE3) strain in the presence of the helper plasmid pSBET
(Schenk et al., 1995). 2.6 l LB cultures were grown at 310 K and
induced in the exponential phase by the addition of 100 mM isopropyl
?-d-1-thiogalactopyranoside (IPTG). After 4 h induction, the
bacteria were pelleted by centrifugation for 15 min at 5000g and
resuspended in buffer A (30 mM Tris–HCl pH 8.0, 1 mM EDTA,
200 mM NaCl). Bacteria were sonicated and centrifuged for 1 h at
16 000g to eliminate insoluble material. The soluble fraction was then
precipitated with ammonium sulfate and the fraction precipitating
between 40 and 80% saturation was collected. The ammonium sulfate
precipitate was redissolved and loaded onto an ACA44 gel-filtration
column (5 ? 75 cm; BioSepra) equilibrated with buffer A. The frac-
tions of interest were pooled, dialyzed against buffer B (buffer A
without NaCl) and loaded onto a DEAE Sephacel column equili-
brated with buffer B. The protein was eluted using a 0–0.4 M NaCl
gradient, dialyzed again using buffer B, concentrated and finally
stored at 243 K for crystallization trials.
2.2. Crystallization strategy
Crystallization conditions for the purified peroxiredoxin domain
were screened using the vapour-diffusion method with a Cartesian
Technology workstation. Sitting drops composed of 200 nl protein
solution at a concentration higher than 5 mg ml?1and 200 nl mother
liquor were equilibrated against 150 ml well solution on a Greiner
plate (Santarsiero et al., 2002). In this study, we used a Greiner
microcrystallization plate with 96-reservoir wells and 288 sitting-drop
circle shelves to perform the screening (Mueller et al., 2001). Initial
screens were performed using the commercially available sparse-
matrix kits Structure Screens 1 and 2 from Molecular Dimensions
Ltd, JBScreens 1–8 from Jena Biosciences and Crystal Screens I and
II from Hampton Research. A Genesis Workstation 150 robot from
Tecan was used to dispense the crystallization solutions (150 ml) in the
The protein was deposited first into all wells, followed by line
dispensing of the crystallizing agent into the 96 wells with an eight-tip
Cartesian robot. The microplate was then manually sealed with a
Hampton Research and incubated at 291 K.
Images of the drops were taken 1, 3, 7 and 30 d after mixing the
protein with the crystallizing agents. We used a Nikon microscope
(Eclipse E600) equipped with a DXM 1200 video camera and an xy
computer driver plate holder. The Lucia version G software (Nikon)
was used to obtain and analyze the images. We obtained crystal-
lization hits in many conditions using ammonium sulfate as a crys-
tallizing agent, especially with JBScreen 6 conditions A2 and C4.
Crystals obtained from drops set up using the Cartesian robot were
small or in needle-cluster form. In order to increase their quality and
size, they were reproduced manually using the hanging-drop vapour-
diffusion method in Linbro 24-well plates. We also tested the effect of
varying the crystallizing agent or protein concentration, pH and drop
volume (McPherson, 1999).
The best crystals were obtained by mixing 1.5 ml 24.8 mg ml?1
protein solution in 30 mM Tris–HCl pH 8.0, 1 mM EDTAwith 1.5 ml
reservoir solution containing 1.8 M ammonium sulfate, 0.1 M Tris–
HCl pH 8.5, 7.5%(v/v) ethylene glycol. The crystals grew within a
week at 291 K.
2.3. Collection and processing of diffraction data
Crystals were prepared for cryocrystallography by transferring
them to reservoir solution containing 20%(v/v) glycerol. A single
crystal was picked up with a cryo-loop (Hampton Research) and
flash-cooled to 100 K in a nitrogen-gas stream (Rodgers, 1994).
Diffraction data were collected on beamline ID14-1 at the
European Synchrotron Radiation Facility. 1?oscillation photographs
were collected. X-ray data were indexed and integrated using
MOSFLM v.6.2.6 for image plates and CCDs (Leslie, 1992). Inten-
sities were further converted to amplitudes and scaled using
programs from the CCP4 suite v.6.0 (Collaborative Computational
Project, Number 4, 1994; Evans, 1997).
Barbey et al.
? Peroxiredoxin domain of natural hybrid protein
Acta Cryst. (2008). F64, 29–31
Crystals of the peroxiredoxin domain. The scale bar is 0.1 mm in length.
3. Results and discussion
Crystals grew as small cubes as shown in Fig. 1. The dimensions of the
crystal used for the final data collection were 200 ? 200 ? 300 mm.
From the symmetry of the diffraction data set and from the absence
of systematic extinctions along the fourfold axis, the space group was
determined to be I422, with unit-cell parameters a = b = 176.67,
c = 141.20 A˚, ?= ? = ? = 90?. The crystal showed ordered diffraction
to 2.9 A˚resolution, with a mosaicity of around 0.5?. Details of the
data-collection statistics are reported in Table 1. The symmetry of the
crystal implies that at least six and possibly as many as eight mole-
cules are present in the asymmetric unit; these would correspond to
51 or 35% solvent content, respectively. Preliminary calculations
deduced from the self-rotation function (POLARRFN; Collaborative
Computational Project, Number 4, 1994) calculated using standard
parameters emphasize significant noncrystallographic axes: twofold
and threefold axes (peaks that reach a maximum of 37% of the
normalized value of the crystallographic fourfold axis appear at
coordinates ! = 45, ’ = 90, ? = 180?and ! = 55, ’ = 45, ? = 120?) are
found in agreement with a trimer of homodimers, but the presence of
a fourfold axis (! = 90, ’ = 0, ? = 90?) corresponding to an octamer
solution is also indicated. Molecular-replacement trials using the
program AMoRe (Navaza, 1994) are currently in progress and are
being performed using various Prx structures available in the PDB as
search models. To date, a large number of peroxiredoxin structures
(43) have been deposited in the PDB. Most of them are of typical
2-Cys peroxiredoxins, 1-Cys peroxiredoxins and type II peroxi-
redoxins. Yeast nuclear thiol peroxidase (PDB code 2a4v), Plasmo-
dium falciparum mitochondrial 2-Cys peroxiredoxin (PDB code
2c0d) and Aeropyrum pernix K1 peroxiredoxin (PDB codes 2cx3 and
2cx4) exhibit the largest amino-acid sequence identity to the
T. maritima peroxiredoxin module described here (30–31%; Altschul
et al., 1997), but only the yeast and A. pernix proteins belong to the
Prx Q/Prx BCP family. In addition, these two proteins possess the two
highly conserved cysteines that are present in approximately half of
the Prx BCP family members, whereas the Prx domain of the
T. maritima hybrid protein displays only the peroxidatic cysteine and
no putative resolving cysteines. The regeneration mechanism used by
this Prx is thus presently unknown and as there is no identified
thioredoxin (the usual reductant for Prx Q and BCP) in the genome
of T. maritima, it is expected that this structural study should provide
data that will lead to a better understanding of the function and
regeneration of these enzymes. Last but not least, this Prx module is
associated with a nitroreductase module and whether and how the
two modules interact in catalysis is still uncertain, especially as the
structure of the full-length protein is not available. The low identity
between the Prx domain of T. maritima and these models, together
with the large number of independent molecules to place, have so far
prevented an unambiguous identification of the molecular-replace-
ment solution. Different modifications of the coordinates (i.e. the
replacement of the non-aligned residues or of all the residues by
alanines or the use of a poly-C?model) and the use of oligomeric
models are currently being performed in order to determine the
phases by molecular replacement.
We acknowledge the European Synchrotron Radiation Facility for
providing access to synchrotron radiation and would like to thank
Emanuela Fioravanti for assistance in using beamline ID14-1,
Philippe Benas and Thierry Prange ´ for their help during data
collection and processing, and Jorge Navaza for access to novel
features of the AMoRe program.
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Acta Cryst. (2008). F64, 29–31 Barbey et al.
? Peroxiredoxin domain of natural hybrid protein
Values in parentheses are for the highest resolution shell.
Unit-cell parameters (A˚)
Resolution range (A˚)
ID14-1, ESRF, France
a = b = 176.67, c = 141.20