Biochemical and thermodynamic comparison of the selenocysteine containing and non-containing thioredoxin glutathione reductase of Fasciola gigantica

Article abstract of DOI:10.1016/j.bbagen.2018.03.007

The thiol-disulfide redox metabolism in platyhelminth parasites depends entirely on a single selenocysteine (Sec) containing flavoenzyme, thioredoxin glutathione reductase (TGR) that links the classical thioredoxin (Trx) and glutathione (GSH) systems. In the present study, we investigated the catalytic and structural properties of different variants of Fasciola gigantica TGR to understand the role of Sec. The recombinant full-length Sec containing TGR (FgTGRsec), TGR without Sec (FgTGR) and TGRsec without the N-terminal glutaredoxin (Grx) domain (?NTD-FgTGRsec) were purified to homogeneity. Biochemical studies revealed that Sec597 is responsible for higher thioredoxin reductase (TrxR) and glutathione reductase (GR) activity of FgTGRsec. The N-terminal Grx domain was found to positively regulate the DTNB-based TrxR activity of FgTGRsec. The FgTGRsec was highly sensitive to inhibition by auranofin (AF). The structure of FgTGR was modeled, and the inhibitor AF was docked, and binding sites were identified. Unfolding studies suggest that all three proteins are highly cooperative molecules since during GdnHCl-induced denaturation, a monophasic unfolding of the proteins without stabilization of any intermediate is observed. The Cm for GdnHCl induced unfolding of FgTGR was higher than FgTGRsec and ?NTD-FgTGRsec suggesting that FgTGR without Sec was more stable in solution than the other protein variants. The free energy of stabilization for the proteins was also determined. To our knowledge, this is also the first report on unfolding and stability analysis of any TGR.

Full text of DOI:10.1016/j.bbagen.2018.03.007

Accepted Manuscript
Biochemical and thermodynamic comparison of the
selenocysteine containing and non-containing thioredoxin
glutathione reductase of Fasciola gigantica
Parismita Kalita, Harish Shukla, Rohit Shukla, Timir Tripathi
PII:
S0304-4165(18)30063-1
doi:10.1016/j.bbagen.2018.03.007
BBAGEN 29058
DOI:
Reference:
To appear in:
Received date:
Revised date:
Accepted date:
14 November 2017
2 March 2018
6 March 2018
Please cite this article as: Parismita Kalita, Harish Shukla, Rohit Shukla, Timir Tripathi
Biochemical and thermodynamic comparison of the selenocysteine containing and non-
,
containing thioredoxin glutathione reductase of Fasciola gigantica. The address for the
corresponding author was captured as affiliation for all authors. Please check if
appropriate. Bbagen(2018), doi:10.1016/j.bbagen.2018.03.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.

ACCEPTED MANUSCRIPT
Biochemical and thermodynamic comparison of the selenocysteine containing and non-
containing thioredoxin glutathione reductase of Fasciola gigantica
Parismita Kalita, Harish Shukla, Rohit Shukla, and Timir Tripathi*
Molecular and Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern
Hill University, Shillong 793022, India
Running title: Comparison of FgTGRsec and FgTGR
*
To whom correspondence should be addressed: Dr. Timir Tripathi, Department of
Biochemistry, North-Eastern Hill University, Shillong- 793022, India. Email:
timir.tripathi@gmail.com, ttripathi@nehu.ac.in. Tel: +91-364-2722141; Fax: +91-364-2550108.
ABSTRACT
The thiol-disulfide redox metabolism in platyhelminth parasites depends entirely on a single
selenocysteine (Sec) containing flavoenzyme, thioredoxin glutathione reductase (TGR) that links
the classical thioredoxin (Trx) and glutathione (GSH) systems. In the present study, we
investigated the catalytic and structural properties of different variants of Fasciola gigantica
TGR to understand the role of Sec. The recombinant full-length Sec containing TGR
(FgTGRsec), TGR without Sec (FgTGR) and TGRsec without the N-terminal glutaredoxin (Grx)
domain (∆NTD-FgTGRsec) were purified to homogeneity. Biochemical studies revealed that
Sec597 is responsible for higher thioredoxin reductase (TrxR) and glutathione reductase (GR)
activity of FgTGRsec. The N-terminal Grx domain was found to positively regulate the DTNB-
based TrxR activity of FgTGRsec. The FgTGRsec was highly sensitive to inhibition by
auranofin (AF). The structure of FgTGR was modeled, and the inhibitor AF was docked, and
binding sites were identified. Unfolding studies suggest that all three proteins are highly
cooperative molecules since during GdnHCl-induced denaturation, a monophasic unfolding of
the proteins without stabilization of any intermediate is observed. The Cm for GdnHCl induced
unfolding of FgTGR was higher than FgTGRsec and ∆NTD-FgTGRsec suggesting that FgTGR
without Sec was more stable in solution than the other protein variants. The free energy of
1

ACCEPTED MANUSCRIPT
stabilization for the proteins was also determined. To our knowledge, this is also the first report
on unfolding and stability analysis of any TGR.
Keywords
Platyhelminthes, parasite, liver fluke, thioredoxin glutathione reductase, glutathione reductase,
thioredoxin reductase, glutathione, activity, homology modeling, auranofin, inhibition
1
. INTRODUCTION
Enzymes associated with redox metabolism encompass the most interesting targets for drug
development against infectious diseases. These enzymes maintain cellular homeostasis and play
a major role in antioxidant defenses [1]. A disturbed redox metabolism results in the alteration of
several important cellular processes that are critical for proper cell function. The redox regulation
is mainly accomplished by oxidative modification of the highly susceptible thiol group (-SH) of
the cysteinyl side chain of proteins [2]. Hence, being crucial players in redox regulation, the
thiol-proteins become the most vulnerable targets of cellular redox imbalance.
All organisms are equipped with a variety of antioxidant thiol-based enzymes that
maintain the cellular redox homeostasis. In most aerobic organisms it is comprised of two
separate redox systems: the glutathione/glutaredoxin (GSH/Grx) system containing Grx, GSH
and NADPH-dependent glutathione reductase (GR); and the thioredoxin (Trx) system containing
Trx and NADPH-dependent thioredoxin reductase (TrxR) [3]. In contrast to these classical redox
systems, the platyhelminth parasites contain a single multifunctional selenocysteine (Sec, U)
containing flavoenzyme, thioredoxin glutathione reductase (TGR), having fused Grx and TrxR
domains that link both these systems [4, 5]. Transcriptomic and genomic data of F. hepatica, T.
solium, Schistosoma sp. and E. multicularis reveals the absence of GR and TrxR genes in these
parasites [6, 7]. TGR is the sole enzyme responsible for both Trx and GSH reduction in parasitic
flukes. In the absence of catalase, the cellular redox homeostasis of these parasites depends
entirely on the TGR, which supplies electrons to both glutathione disulfide (GSSG) and Trx.
Also, TGR is the key enzyme responsible for reactive oxygen species (ROS) detoxification,
redox homeostasis maintenance, and synthesis of deoxyribonucleotides in parasitic flukes [8, 9].
st
Helminth TGR encodes a Sec in its C-terminal end. Sec is the 21 amino acid encoded by the
UGA codon in the mRNA and it acts as an important active site residue in many redox active
2

ACCEPTED MANUSCRIPT
selenoenzymes such as TGR, TrxR, glutathione peroxidase (GPx), iodothyronine deiodinase, etc,
in all forms of life [10, 11]. Its role is also implicated in redox signaling, antioxidant defense,
thyroid hormone metabolism and immune response [11]. Co-translational incorporation of Sec
into the polypeptide chain involves a complex machinery that includes a SECIS element
(selenocysteine insertion sequence), SECIS binding protein (SBP1 and SBP2), a Sec-
[
Ser]Sec
tRNA
, Sec specific elongation factor and other accessory proteins[10]. Several structures
of TGR have been determined [7, 12, 13]. The X-ray crystallographic structure of S. mansoni
TGR shows that like other members of the pyridine nucleotide disulfide oxidoreductase family, it
is a homodimeric protein with monomers arranged in a head-to-tail fashion creating a twisted
‘W’ shape [14]. Each monomer is composed of two key domains: an N-terminal Grx domain and
a C-terminal TrxR domain. The two Grx domains are at the top of the two outer arms of the
dimer ‘W’. In addition to an NADPH binding site, the TrxR domain has two redox active centers
viz. a -CX C- motif in the FAD-binding domain (also found in GR and TrxR) and a Sec
4
containing -GCUG- redox center in the C-terminus. The Grx domain has a GSH binding site and
a redox active Cys pair (-CXXC- or -CXXS-) [15]. The dithiol Grx containing TGR exhibits a
unique hysteresis phenomenon as its GR activity is regulated by the [GSSG]/[GSH] ratio. Under
high GSSG concentrations, the GR activity of TGR decreases and the behavior is reverted in the
presence of GSH, Trx, or DTT. This behavior is associated with glutathionylation of TGR by
GSSG and is blocked by deglutathionylation [16, 17]. In contrast, the monothiol Grx containing
TGR shows no hysteresis for GR activity [18].
Fascioliasis is a major food borne tropical disease caused by the organisms of the genus
Fasciola. It affects over 600 million animals globally resulting in economic losses of >US$3
billion p.a. in livestock production. Around 25–100% of cattle in Africa, the Middle East, and
Southeast Asia are affected annually and in temperate countries it also affects ~70% of humans
[
19]. It is estimated that around 2.4–17 million people are infected and approximately 180
million people are at risk globally [20]. Africa, Europe, the Middle East (including Egypt),
Southeast Asia, and Latin America, are listed as the major human fascioliasis endemic areas,
with Bolivian Altiplano having the highest prevalence [21, 22]. Triclabendazole (TCZ) is the
only drug of choice recommended by WHO against these flukes [23]. Recent reports of TCZ
resistance highlight the need for new chemotherapeutics [24-28]. With the help of antioxidant
enzymes, these parasites can scavenge the reactive species produced as a result of host immune
3

ACCEPTED MANUSCRIPT
response and can survive in the host for longer periods. In recent years, TGR has emerged as a
rational drug target for the treatment of platyhelminthic infections as it is not present in the
human counterparts. Auranofin, an inhibitor of TGR and TrxRs, kills larval worms in vitro and
has been reported to partially cure Schistosoma infection [29, 30]. Besides auranofin, several
other inhibitors have also been identified for TGRs [31-35]. Limited knowledge is available
regarding the structure-function relationship of redox enzymes of parasitic flukes. In the present
study, we have investigated the structure-function relationship, stability and folding analysis of
FgTGRsec and its truncated variants to define the molecular, functional and structural
characteristics of this enzyme.
2
2
. MATERIALS AND METHODS
.1 Materials
The molecular biology kits and Ni-NTA agarose were purchased from Qiagen, CA, USA. The
dNTPs and enzymes were purchased from New England Biolabs, MA, USA. All other reagents
and chemicals were purchased either from Sigma-Aldrich Chemical Company, St. Louis, MO,
USA, or Sisco Research Laboratories, Mumbai, India and were of the highest purity available.
Bacterial culture media was purchased from Himedia Laboratories, Mumbai, India.
2
.2 Collection and identification of parasites
The adult liver flukes were collected from the liver of naturally infected cattle from the local
slaughterhouse at Bada Bazaar, Shillong, India. The flukes were washed extensively with chilled
phosphate buffer saline (pH 7.5). The flukes were identified as F. gigantica using morphological
properties.
2
.3 Isolation of total RNA and cDNA synthesis
The flukes were crushed in a pestle and mortar with liquid nitrogen, and the total RNA was
isolated using the RNeasy mini kit (Qiagen, USA) as per the manufacturer instructions. The first
strand cDNA was synthesized using QuantiTect Reverse Transcriptase kit (Qiagen, USA) as per
the manufacturer instructions.
2
.4 Cloning, overexpression and purification of FgTGRsec and ∆NTD-FgTGRsec
4

ACCEPTED MANUSCRIPT
Fgtgrsec and ∆NTD-Fgtgrsec were amplified by PCR using primers containing a bacterial type
SECIS element for Sec insertion. Three sets of primers were designed based on the full length
TGR sequence of F. gigantica (Supplementary Table 1). The reverse primer of both FgTGRsec
and ∆NTD-FgTGRsec contained a bacterial type SECIS for Sec insertion, whereas that of
FgTGR was designed without a bacterial-type SECIS. The stop codon of the gene was omitted in
the reverse primers and the resulting peptide ends at stop codon coded by the SECIS element.
+
The amplicons were cloned into pSK+ vector and further sub-cloned to pET23a(+) (Amp ) and
+
pET28a(+) (Kan ) vectors, respectively. The method described by Kuntz et al. was followed for
expression of the recombinant selenoproteins [7]. The recombinant plasmids were transformed
into competent E. coli BL21 (DE3) expression host cells and grown in Luria-Bertani media
containing respective antibiotics at 37 °C until an A600 ~0.7 was achieved and then induced at 20
°
C with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). Also, as a source of selenium, 5
µM Na SeO was added to the induced culture and 10% glycerol was added for enhancing
2
3
solubility and to ensure slow growth. Induced cultures were further grown for ~12 h with
continuous shaking at 150 rpm and 20 °C. After sonication, the clear cell lysate was loaded onto
a Ni-NTA column pre equilibrated with 50 mM phosphate buffer (pH 8.0), containing 300 mM
NaCl and washed with the same buffer to remove unbound contaminants. The column was
washed with increasing concentrations of imidazole, and the proteins were eluted with
equilibration buffer containing 300 mM imidazole. The purified proteins were dialyzed against
2
0 mM phosphate buffer (pH 8.0), containing 150 mM NaCl. Overexpression and purification of
truncated FgTGR were performed as mentioned earlier [36]. The purity and the molecular weight
of the recombinant protein were determined using 12% SDS-PAGE. Quantitation of the
recombinant proteins was carried out using the Bradford method using bovine serum albumin as
the standard.
2
.5 Size exclusion chromatography (SEC)
SEC experiments were carried out on a Superdex 200 10/300 GL column on an AKTA FPLC
GE Healthcare Biosciences, USA) pre-calibrated with standard molecular weight markers. The
TM
(
column was equilibrated and run with 20 mM phosphate buffer (pH 8.0), containing 150 mM
NaCl at 25 °C at a flow rate of 0.4 mL/min with detection at 280 nm and 450 nm for detection of
protein and bound FAD respectively.
5

ACCEPTED MANUSCRIPT
2
.6 Biochemical assays
TrxR and GR activities of FgTGRsec, FgTGR and ∆NTD-FgTGRsec were analyzed in a Cary 50
Varian) spectrophotometer at 25 °C based on the methods described by Kuntz et al.[7]. The
(
TrxR activity was determined by the 5,5′-diothiobis(2-nitrobenzoic acid) (DTNB) reduction
assay. The assay solution contained 1 µM recombinant enzyme, 200 µM NADPH and 10 mM
EDTA in 20 mM phosphate buffer (pH 7.2), containing 150 mM NaCl. The DTNB concentration
was varied from 0.5 mM to 5 mM. The increase in absorbance at 412 nm during the first three
−
1
−1
min was monitored, and the extinction coefficient of 13.6 mM cm for 5-thionitrobenzoic acid
(TNB) was used for calculation of activity.
The GR activity was determined using GSSG. The reaction mixture contained 1 µM
recombinant enzyme, 200 µM NADPH and 1 mM EDTA in 20 mM phosphate buffer (pH 7.2)
and 150 mM NaCl. The GSSG concentration was varied from 0.5 µM to 5 µM. The decrease in
NADPH consumption at 340 nm during the first three min was monitored, and the extinction
−
1
−1
coefficient of 6.22 mM cm for NADPH was used for calculation of activity.
2
.7 Inhibition studies
To assess the effect of the inhibitor auranofin (AF) on FgTGRsec, incubation mixtures were
prepared containing 3 µM FgTGRsec with and without AF (0.5 to 5 µM). For DTNB-based
TrxR activity inhibition assay, reaction mixtures were prepared as mentioned above. The
measurements were performed at 120 min after incubation. The reaction was initiated by
addition of 5 mM DTNB and followed by the increase in absorbance at 412 nm.
For GR activity inhibition assay, reaction mixtures were prepared as mentioned above.
The measurements were performed at 120 min after incubation. The reaction was initiated by
addition of 500 µM GSSG and followed by the decrease in absorbance at 340 nm.
2
.8 Fluorescence quenching with substrates and inhibitor
For fluorescence quenching experiments, the stock solutions of the quenchers - DTNB (5 mM),
GSSG (10 mM) and AF (250 µM) were prepared separately. Each quencher was added to
achieve the desired range of quencher concentration in 1 µM protein sample. The samples were
excited at 280 nm and the emission spectra were recorded in the range of 300–500 nm. The
6

ACCEPTED MANUSCRIPT
Stern-Volmer quenching constants were calculated using the plot of F /F values against the
0
1
input concentration of quenchers, in the Stern-Volmer equation,
F /F = 1 + K [Q]
0
1
SV
where F and F are the fluorescence intensities at a particular wavelength in the absence and
0
presence of quencher, respectively, Ksv is the Stern-Volmer constant, and [Q] is the
concentration of the quencher.
2
.9 Equilibrium unfolding experiments
A stock solution of 8 M GdnHCl was prepared in 20 mM phosphate buffer (pH 7.2) containing
50 mM NaCl. All samples were incubated for 4 h to achieve denaturation equilibrium before
the readings were taken.
1
2
.10 Fluorescence and circular dichroism (CD) spectroscopy
Fluorescence spectra were recorded with Perkin-Elmer Life Sciences LS55 fluorescence
spectrometer in a 10 mm path length quartz cuvette. Protein concentration used was l µM for all
experiments and the measurements were carried out at 25 ºC. Slit width for both emission and
excitation were kept as 10 nm. For monitoring Trp and FAD fluorescence, the excitation
wavelength of 285 nm and 445 nm, respectively, were used and the spectra were recorded
between 300-500 nm and 450-600 nm, respectively.
The CD measurements were made on JASCO J1500 spectropolarimeter calibrated with
ammonium (β)-10-camphorsulfonate with a 5 mm path length cell at 25 °C at a protein
concentration of 1 µM. Three consecutive scans were accumulated and an average spectra was
stored. The values obtained were normalized by subtracting the baseline recorded for the buffer
under similar conditions.
2
.11 Calculation of free energy of stabilization
Following a two-state model of unfolding of a protein, the CD and fluorescence spectroscopic
data were converted into the free energy of unfolding for each data point (ΔG ). The ΔG values
D
D
were then plotted against [GdnHCl] to determine the free energy of stabilization in the absence
H20
of denaturants (ΔGD ) according to the linear extrapolation method [37] as described earlier
[
38-41].
7

ACCEPTED MANUSCRIPT
2
.12 Homology modeling and validation
Homology modeling was performed using Modeller 9.16 [42]. Initially, the BLAST
http://blast.ncbi.nlm.nih.gov/Blast.cgi) search was performed to find the closely related
(
homologs of FgTGRsec and then template was selected on the basis of identity, query coverage
and structure resolution. The structure of S. japonicum TGR (SjTGR) (PDB ID: 4LA1) [12] was
taken as the template to model the dimeric FgTGRsec. The obtained structure was superimposed
with the model to find the reliability using Chimera 1.10.2 [43]. Modeled structure was validated
using ProSA [44], PROCHECK [45] and Verify 3D server [46]. The secondary structure was
predicted by using SOPMA tool.
2
.13 Molecular docking
Molecular docking of FgTGRsec with AF was performed by using Molegro Virtual Docker
MVD) as earlier [47, 48]. The structure of AF and TP gold moiety were drawn using Chemdraw
followed by energy minimization of ligand structures by Amberff99SB force field using Chimera
.10.2. Prior to docking, the protein structure was prepared using MVD [49]. The best pose
(
1
having the energetically favorable structure was selected. MVD calculated the interaction
energies between ligands and protein. The MolDock Score was used for energy calculation. The
residues defined in previous studies were selected for molecular docking [31]. The cavity used
for virtual screening had the size constraint of 15 Å. The docking parameters for virtual
screening included the number of runs (100), population size (50), max iterations (2,000), scaling
factor (0.50), and crossover rate (0.90). Ten binding pose were generated by molecular docking
and selected on the basis of binding affinity as well as best binding conformation.
3
. RESULTS
3
.1 Purification of FgTGRsec, FgTGR and ∆NTD-FgTGRsec
The full-length Sec containing TGR (FgTGRsec), TGR without Sec and the last amino acid
FgTGR) and TGR without the N-terminal Grx domain (∆NTD-FgTGRsec) were purified as
described above. The purified proteins were homogenous as indicated by single protein bands of
66 kDa for both FgTGRsec and FgTGR; and ~54 kDa for ∆NTD-FgTGRsec, respectively on
(
~
SDS-PAGE (Fig. 1A). The molecular mass of purified recombinant FgTGRsec, FgTGR and
8

ACCEPTED MANUSCRIPT
∆
NTD-FgTGRsec were determined under non-dissociating conditions by size-exclusion
TM
chromatography (SEC). Gel filtration of the recombinant proteins on Superdex S-200 column
calibrated with various molecular weight standards showed a single peak for each protein at 280
nm with retention volumes of 13.70 mL, 13.65 mL and 14.15 mL, respectively (Fig. 1B). The
deep yellow color of the proteins indicated the presence of bound FAD that was confirmed
through the 450 nm absorbance peaks for all proteins in SEC (data not shown). When the elution
volumes of the marker proteins were plotted as a function of log of molecular weight, the
FgTGRsec, FgTGR and ∆NTD-FgTGRsec were found to have molecular weights of about 132
kDa, 132 kDa and 108 kDa, respectively.
3
.2 Structural characteristics of the recombinant proteins
The Trp fluorescence spectra of FgTGRsec, FgTGR and ∆NTD-FgTGRsec are shown in Figure
A. An emission wavelength of about 336 nm, 334 nm and 340 nm was observed for the
2
recombinant FgTGRsec, FgTGR and ∆NTD-FgTGRsec, respectively. The sequences of
FgTGRsec, FgTGR and ∆NTD-FgTGRsec encode 9, 9 and 8 Trp residues, respectively. In
general, the buried Trp residues in folded protein show fluorescence emission maxima between
3
30-335 nm [50, 51]. Thus the Trp residues in FgTGRsec and FgTGR are predominantly buried
in the hydrophobic core. The analysis of far-UV CD spectra demonstrates the presence of α/β
type secondary structure (Fig. 2B) in all the three proteins. From these results, it is evident that
all these proteins are present in the properly folded conformation.
3
.3 Sec597 is responsible for higher DTNB-based TrxR activity in FgTGRsec
Recombinant FgTGRsec encodes a Sec at position 597. A decrease in fluorescence intensity was
observed following the binding of DTNB with FgTGRsec and FgTGR (Supplementary Fig. 1).
The extent of quenching was greater in the case of FgTGRsec (~300% decrease from the starting
point) than the FgTGR (~ 200% decrease from the start). The comparative larger reduction in the
intrinsic fluorescence of FgTGRsec observed upon DTNB binding with respect to FgTGR
suggests that FgTGRsec may have a greater affinity for DTNB than FgTGR. The fluorescence
-3
data were further analyzed using the Stern–Volmer plot. Higher Ksv of FgTGRsec (60.6 x 10
-1
-3
-
µM ) confirmed the higher affinity of DTNB towards FgTGRsec than FgTGR (42.5 x 10 µM
1
)
(Fig. 3A and Table 1).
9

ACCEPTED MANUSCRIPT
We then performed comparative enzymatic activity analysis of FgTGRsec and FgTGR
with DTNB. It was observed that FgTGRsec showed higher activity than FgTGR (Fig. 3B). The
result of activity analysis is in corroboration with the binding profile of DTNB with FgTGRsec
and FgTGR. The decrease in activity of FgTGR may be attributed to the absence of Sec at the C-
terminal region of the protein that is necessary to attain optimal catalytic behavior.
3
.4 FgTGRsec has higher GR activity with respect to FgTGR
The N-terminal Grx domain of TGR is also involved in the GR activity. Studies suggest that
there are two different binding sites for GSSG, one at the Grx domain and another in the TrxR
domain. Also, some TGR truncations lacking Grx domain were found incapable of reducing
GSSG [52-54]. ∆NTD-FgTGRsec also showed almost no/negligible GR activity in absence of
Grx domain (data not shown). Moreover, Grx domain contacts with the N-terminal part of the
TrxR domain, thus allowing a shorter route of the electron transfer [55]. To characterize this
activity, GSSG was used as a substrate. For comparative analysis of the binding of GSSG with
FgTGRsec and FgTGR, fluorescence quenching analysis was performed. A decrease in the
quantum yield of fluorescence with increasing concentration of GSSG represents the binding of
GSSG with the protein. The fractional change in quenching was found to be greater for
FgTGRsec than FgTGR (Supplementary Fig. 2). Stern-Volmer analysis of quenching experiment
-3
-1
suggested that the binding affinity of GSSG with FgTGRsec (Ksv = 0.7 x 10 µM ) is greater
-3
-1
than that of FgTGR (Ksv = 0.5 x 10 µM ) (Fig. 3C and Table 1). This implies that FgTGRsec
may have higher GR activity than FgTGR due to favorable GSSG binding. To confirm this, we
performed the GR activity analysis of FgTGRsec and FgTGR with GSSG and observed that
FgTGRsec indeed shows higher GR activity than FgTGR (Fig. 3D and Table 2).
3
.5 The N-terminal Grx domain positively regulates the DTNB-based TrxR activity of
FgTGRsec
To examine the possible role of the N-terminal Grx domain in the DTNB-based TrxR activity of
FgTGRsec, we constructed a truncation for FgTGRsec lacking the N-terminal Grx domain
(residues 1 to 109) (∆NTD-FgTGRsec). The structural feature of the ∆NTD-FgTGRsec has been
discussed in section 2.2. To analyze the effect of Grx domain on the DTNB-based TrxR activity
of the proteins, we performed two sets of experiments. Firstly, we determined the comparative
1
0

ACCEPTED MANUSCRIPT
binding analysis of DTNB with FgTGRsec and ∆NTD-FgTGRsec using fluorescence quenching.
Stern-Volmer analysis of quenching experiment suggested that the binding affinity of DTNB to
-3
-1
∆
NTD-FgTGRsec (Ksv = 52.2 x 10 µM ) was slightly less than that of full length FgTGRsec
-3
-1
(
Ksv = 60.6 x 10 µM ) (Fig. 4A and Table 1). The extent of quenching was greater in
FgTGRsec than ∆NTD-FgTGRsec (Supplementary Fig. 3), which suggests that the binding of
DTNB was partly compromised in the ∆NTD-FgTGRsec. To confirm whether this binding
pattern correlates with the activity, we performed activity assay and found that ∆NTD-
FgTGRsec exhibited about two-fold lesser DTNB-based TrxR activity when compared with the
FgTGRsec (Fig. 4B and Table 2). These results suggest that the N-terminal Grx domain has a
positive role in regulating TrxR activity.
3
.6 Binding and inhibition of catalytic activity of FgTGRsec by AF
AF is a gold-containing compound that binds to and inhibits TGRsec activity of several parasitic
flukes [13, 17, 30, 56, 57]. To evaluate the effect of AF on the activity of FgTGRsec, inhibition
studies were performed. Stern-Volmer analysis of quenching experiment shows strong binding
-3
-1
affinity of AF with FgTGRsec (Ksv = 58.8 x 10 µM ) (Supplementary Fig. 4) (Fig. 5A). AF
inactivates the activity of FgTGRsec in a concentration dependent manner (Fig. 5B) with an IC50
of 0.87 µM for DTNB-based TrxR activity and 8.30 µM for GR activity (Fig.5C). Both TrxR
and GR activities were found to be decreased by ~10 fold at ~2 µM concentration of AF at a
fixed concentration of DTNB and GSSG, respectively.
3
.7 Homology modeling, energy minimization and structure validation of FgTGR
The crystal structure of FgTGRsec is not available so we modelled the structure of FgTGR. The
sequence was submitted to PDB BLAST for predicting homolog structures. The crystal structure
of SjTGR (PDB ID: 4LA1) was selected as a template for modeling the FgTGR structure. We
found 99% query coverage with 63% sequence identity in pairwise sequence alignment. The
predicted structure was modified with the Sec residue. The structure alignment between SjTGR
and FgTGRsec was carried out to predict the reliability of the modelled structure. The RMSD
value was found to be 0.277 Å which indicates that the predicted model is very close and similar
to the template structure. The sequence alignment and structure alignment of FgTGRsec with
SjTGR is shown in supplementary Figure 5 and 6. The structure was further validated by using
1
1

ACCEPTED MANUSCRIPT
Ramachandran plot, Z score and Verify-3D score. Ramachandran plot showed that most of the
amino acids were present in most favored region with 5.8% in additional allowed region and
only three residues (263, 519 and 521) belong to the disallowed region (Supplementary Fig. 7).
The energy window of FgTGRsec model showed that only some of the C-terminal residues lie in
the positive window while all other residues belong to the negative window (Supplementary Fig.
8
1
) indicating that the overall energy of the model is good. The Z-score of the predicted model (-
2.25) was similar to the template structure (-12.41) suggesting that the accuracy of predicted
model is good (Supplementary Fig. 9). The verify-3D showed a score of 95.48% for FgTGRsec
that suggests the good quality of the model (Supplementary Fig. 10). All the stereo-chemical
parameters suggested that the predicted model was good and can be used for further analysis.
The stereochemical parameters of FgTGRsec were found to be the same as our previous study
with FgTGR model [31]. The SOPMA server was used to predict the secondary structure content
of FgTGRsec. The percentage of α-helix, extended strand, β-turn and random coil were found to
be 31.44%, 25.08%, 10.70% and 32.78% respectively.
3
.8 Molecular docking
AF was docked against FgTGRsec to predict the binding pattern. It showed binding with several
catalytic residues with a good docking score. AF showed -141.32 MolDock Score with five
hydrogen bonds. The Sec597 from chain A and Lys125, Gln216 from chain B were involved in
hydrogen bonding with FgTGRsec. Sec597 form a hydrogen bond between the N-atom of the AF
and its own amide group. The amide group of catalytically important polar amino acid Lys125
interacts with the O-atom of the ligand by hydrogen bond. Gln216 form three hydrogen bonds
with the ligand. The amide group of the polar amino acid Gln216 interacts with the three O-atom
of the ligand. The residues Ala595, Thr594, Sec597, Thr580, Ser504, Glu576 and Val593 from
chain A and Leu209, Tyr213, Gln216, Lys125, Arg451, Ile447 from chain B are involved in
hydrophobic interactions (Fig. 6A). AF interacts with both the chains and also with the
catalytically important amino acid residues.
3
.9 Triethylphosphine-gold moiety (TP gold)
A crystal structure of TGR in complex with gold has been reported by Angelucci et.al [56]. In
this structure, they co-crystallized AF with SmTGR and predicted the binding of gold to the
1
2

ACCEPTED MANUSCRIPT
protein. They found that gold binds at three places. We also docked a single TP gold moiety to
FgTGRsec to predict its binding pattern. We found that it shows a MolDock Score of -52.57 that
is less than AF. This is due to the larger size of AF that leads to higher binding affinity. The TP
gold moiety did not form any hydrogen bond with the protein though it showed hydrophobic
interactions with residues- Phe506, Pro508, Leu509, His571, Pro572, Glu576 and Cys574.
Interestingly, during the gold binding, the position of the gold moiety was changed, and it
showed very close interaction with a catalytically important amino acid His571 with a distance
of 3.847 Å (Fig. 6B).
3
.10 GdnHCl-induced changes in structural and functional properties of FgTGRsec and
FgTGR
To study the difference in the folding behavior and conformational stability of FgTGRsec and
FgTGR, GdnHCl-induced unfolding of both the proteins was studied. Figure 7A summarizes the
changes in Trp emission maxima of both FgTGRsec and FgTGR with increasing concentrations
of GdnHCl. A sigmoidal loss in the emission maxima of FgTGRsec and FgTGR from 336 nm
and 334 nm, respectively to about 356 nm was observed between 0 and 4 M GdnHCl. At
GdnHCl above 4.0 M, the emission maxima reached 356 nm demonstrating that the proteins are
completely unfolded under these conditions as the exposed Trp residues in an unfolded protein
show emission maximum between 350-356 nm [51].To test whether the two-state unfolding of
FgTGRsec and FgTGR reflects the disruption of the overall structure of the proteins or is merely
an indicative of local unfolding near the Trp microenvironment, we studied the GdnHCl-induced
alterations in the secondary structure of the proteins. Figure 7A also summarizes the effect of
increasing concentrations of GdnHCl on the CD ellipticity at 222 nm for FgTGRsec and FgTGR.
Increase in GdnHCl concentrations between 0.5 and 3.0 M GdnHCl resulted in a gradual
decrease in CD ellipticity at 222 nm of FgTGRsec, and almost complete loss of CD signal was
observed at about 3.5 M GdnHCl. For FgTGR complete loss of CD signal was observed at about
4
.0 M GdnHCl. Thus, the far-UV CD results show that FgTGRsec unfolded at a lower GdnHCl
concentration than FgTGR, which was similar to that observed from changes in tryptophan
fluorescence. The average Cm calculated from fluorescence and CD signal of FgTGRsec was
found to be ~2.1 M while that of FgTGR was observed at ~2.4 M (Table 2).
1
3

ACCEPTED MANUSCRIPT
FgTGRsec contains a single, tightly but non-covalently bound FAD molecule and nine
Trp residues in each subunit. In the structure of FgTGRsec, the FAD moiety is present towards
the interior of the protein core. It is well known that a strong correlation between alterations in
enzymatic activity and FAD fluorescence polarization exists in flavoproteins. Hence, we
monitored the changes in FAD fluorescence polarization of FgTGRsec and FgTGR at increasing
GdnHCl concentrations. A sharp decrease in the fluorescence polarization and the GR activity of
FgTGRsec and FgTGR was observed between 0 and 1 M GdnHCl and beyond this concentration
a complete loss of activity was observed (Fig. 7B). This suggests a slight perturbation in the Grx
domain active site that is involved in GR activity of the protein. The DTNB-based TrxR activity
also showed gradual decrease between 0 and 2 M GdnHCl. Beyond this denaturant concentration
a complete loss of polarization as well as activity occurs. Interestingly, both FgTGRsec and
FgTGR showed an initial increase in the DTNB-based TrxR activity at 0.25 M and 0.5 M
GdnHCl concentration that may be due to slight melting of the secondary structure making the
active site more accessible to substrate (DTNB) binding. At these concentrations, even though
FgTGR showed ~2 fold higher activity than the FgTGRsec, its activity decreased abruptly
beyond 1.0 M GdnHCl while complete loss of DTNB-based TrxR activity of FgTGRsec was
seen beyond 2.5 M (Fig.7C).
We further determined the conformational stability of FgTGRsec and FgTGR assuming
the two-states model of unfolding. The GdnHCl-induced unfolding curves of both fluorescence
and far-UV CD experiments were used to determine the free energy of stabilization in the
H2O
absence of denaturants (ΔGD ) by linear extrapolation of the ΔG values to zero denaturant
D
H20
concentration (Fig. 7D). From these measurements, a comparable ΔGD value for FgTGRsec
−
1
−1
was calculated to be around 16.32 kcal.mol and 16.24 kcal.mol , respectively. Similarly, the
H20
−1
−1
ΔGD value for FgTGR was calculated to be around 17.96 kcal.mol and 17.35 kcal.mol ,
respectively.
3
.11 Comparison of GdnHCl-induced changes in structural and functional properties of
FgTGRsec and ∆NTD-FgTGRsec
To understand the effect of the N-terminal Grx domain in the unfolding of TGR, GdnHCl
induced unfolding of FgTGRsec and ∆NTD-FgTGRsec was studied. GdnHCl induced unfolding
of ∆NTD-FgTGRsec also showed cooperative transitions in both fluorescence and far-UV CD
1
4

ACCEPTED MANUSCRIPT
spectroscopy (Fig. 8A). These results showed a sigmoidal loss of structure in both proteins
leading to the complete loss in structure beyond 3.5 M GdnHCl. The correlation between FAD
polarization and its DTNB-based TrxR activity was also monitored for ∆NTD-FgTGRsec. A
significant decrease in FAD polarization and activity was observed between 0 and 1.0 M
GdnHCl concentration (Fig. 8B). Similar to that of the full-length FgTGRsec, ∆NTD-FgTGRsec
also showed an initial increase in its activity between 0.25 to 0.5 M GdnHCl, but beyond 1.0 M
GdnHCl, complete loss of activity was observed. Also, a lower Cm value of ∆NTD-FgTGRsec
(~1.9 M) suggests that it is comparatively less stable than the FgTGRsec (~2.1 M) (Table 2).
H20
From these measurements, a comparable ΔGD
value for ∆NTD-FgTGRsec was
−
1
−1
calculated to be around 15.98 kcal.mol and 15.90 kcal.mol , respectively, which is slightly less
than that of the full-length FgTGRsec, suggesting a relatively feasible thermodynamic process
for ∆NTD-FgTGRsec unfolding by GdnHCl (Fig. 8C).
4
. DISCUSSION
The redox homeostasis of parasites is precarious due to the oxidative stress imposed by the host,
and in the case of parasitic helminths, it is absolutely dependent on selenium. This has led to
successful targeting of TGR in these parasites. Parasitic flukes have a unique and simplified
thiol-based redox system. It represents an unusual fusion of the conventional Trx and GSH
system, where a single enzyme TGR supplies electrons to both Trx and GSSG. TGR was first
discovered in S. mansoni [53] and since then, it has been reported in a number of parasitic flukes,
including S. japonicum, F. hepatica, and F. gigantica [16, 36, 58, 59]. TGR is also present in
mammals, albeit in low amounts. It is expressed in male germ cells and found only at low levels
in other tissues [54, 60]. Parasitic flukes lack catalase, and in the absence of catalase, the thiol
redox homeostasis of these parasites depends completely on TGR. TGR is the main enzyme
responsible for the maintenance of redox homeostasis, ROS detoxification, and
deoxyribonucleotides synthesis in these organisms. Unlike GR, TrxR and TGR enzymes contain
a C-terminal extension of 16 amino acids, where an additional active site (-GCUG-) is located
and is indispensable for most functions of these enzymes. TGR is characterized as
selenoenzymes, with Sec occurring as the penultimate amino acid in the redox center -GCUG-.
Selenium has multifunctional importance in selenoenzymes. It is a homolog of Cys with the thiol
chain replaced by selenol moiety. We propose that, Sec is more nucleophilic than sulfur, and
1
5

ACCEPTED MANUSCRIPT
therefore, its presence may increase the reaction rate of Sec-containing enzymes. Though it has
been shown that replacement of sulfur with selenium in a thiol/disulfide exchange reaction
accelerates the rate of reaction whether selenium acts as a nucleophile, electrophile, or leaving
group [61]. Additionally, after reacting with substrates, the resultant thiol-selenolate may
efficiently accept electrons from other redox-active Cys pairs as Sec has a lower pKa value than
Cys, making it a better leaving group. In addition, under the conditions of oxidative stress,
selenoenzymes are more resistant to inactivation by irreversible oxidation than Cys-containing
enzymes.
In the present study, we studied the catalytic and structural properties of different variants
of Fasciola gigantica TGR to understand the role of Sec. The ORF of FgTGR was fused with a
bacterial type SECIS element by PCR to form a chimeric gene. We constructed three enzyme
forms- full-length Sec containing TGR (FgTGRsec), TGR without Sec and the last amino acid
(FgTGR) and TGR without the N-terminal Grx domain (∆NTD-FgTGRsec). All the three
proteins were purified, and the SEC results suggest that all three proteins exist as dimers in
solution and that the TrxR domain is responsible for dimerization of the subunits. We further
examined the structural properties of the proteins and found the proteins to be in a properly
folded conformation. A lower value of fluorescence emission maxima for FgTGR relative to
FgTGRsec implies slight compactness of the protein without a Sec tail end. The ∆NTD-
FgTGRsec shows comparatively higher fluorescence emission maxima relative to both
FgTGRsec and FgTGR. This may be due to the absence of the Grx domain that leads to slight
exposure of certain Trp residues towards solvent.
We observed that FgTGRsec shows higher DTNB-based TrxR and GR activity than
FgTGR. This property of the enzyme may be attributed to the presence of the Sec. The C-
terminal selenotetrapeptide of TGR is known to shuttle electrons from NADPH to both Trx and
GSSG [11]. Also, at physiological pH, the selenol of Sec is mainly in its anionic (pKa 5.2) form.
This lower pKa value and stronger nucleophilicity make FgTGRsec more reactive than FgTGR
[
11]. To study the role of the Grx domain in the DTNB-based TrxR activity of FgTGRsec, we
performed comparative binding analysis of DTNB with FgTGRsec and ∆NTD-FgTGRsec. The
result suggests that the binding of DTNB was partly compromised in the ∆NTD-FgTGRsec. The
decrease in binding efficiency of DTNB towards ∆NTD-FgTGRsec might be because of some
structural perturbations that occurred due to the absence of the Grx domain. The comparative
1
6

ACCEPTED MANUSCRIPT
activity assays also confirmed this observation. GR activity studies suggested that the Sec was
required for the transfer of electrons from the thiol/disulfide active site of FgTGR to the Grx
domain. Thus, the physiologically relevant NADPH dependent activities of FgTGR were
dependent on Sec. These results suggest that the Sec is responsible for higher DTNB-based TrxR
and GR activity in FgTGRsec and that the N-terminal Grx domain positively regulates DTNB-
based TrxR activity. It was also observed that like other dithiolic Grx domain containing TGRs,
the GR activity of FgTGRsec exhibited a hysteretic kinetic behavior at high GSSG
concentrations (Supplementary Fig. 11).
The structure of FgTGRsec was modeled using the crystal structure of SjTGR as a template.
The RMSD showed an excellent value of 0.277 Å indicating the accuracy of the structure. AF is
a well-known inhibitor for TGR of parasitic flukes [7, 30]. It is a pro-drug that delivers the gold-
containing compound to Sec containing flavoreductases (TrxR and TGR) more effectively than
non-Sec containing ones (GR) where this preference has been attributed to the high affinity of
selenium for gold [62]. Structurally, it has a linear two coordinated Au(I) center with a TP-gold
ligand and a tetraacetylthioglucose ligand (Supplementary Figure 12). The phosphine moiety
confers membrane solubility to AF, whereas the tetraacetylthioglucose ligand is excreted in vivo,
suggesting that the TP-gold is the active therapeutic compound [63]. AF inhibits SmTGR at a
nanomolar concentration (IC ~7 nM) and subsequently reduces worm burden in mice [7, 56].
5
0
We observed that the disulfide reductase activity of FgTGRsec was inhibited by AF, suggesting
that FgTGR is dependent on Sec with an IC of 0.87 µM for DTNB-based TrxR activity and 8.3
5
0
µM for GR activity. Molecular docking studies showed that the binding of AF can change the
conformation of the enzyme and lead to stop the electron flow. His571 is a key catalytic residue
in the formation of selenolate anion that is conserved in human and Plasmodium TrxR and other
TGR homologs from parasitic flukes [14, 64, 65]. The gold can reach and interact with the
His571 residue of FgTGRsec leading to the disruption of the electron transfer mechanism. Such
interaction of gold and His residue of various selenoenzymes have been shown in previous
studies [66, 67] and thus validate our observation.
Lastly, we compared the folding and conformational stabilities of Sec containing and non-
containing TGR and also determined the role of the Grx domain in the folding and stability. The
results were compared with the activity data. To our knowledge, no other investigation to date
has been performed to study the unfolding and stability parameters of any TGR. Our results
1
7

ACCEPTED MANUSCRIPT
indicate that GdnHCl induces cooperative unfolding of the proteins without stabilization of any
intermediate state (Fig. 7A). The Cm of FgTGRsec was found to be lower than that of FgTGR
suggesting that FgTGRsec is comparatively less stable than FgTGR. The Cm value represents
the midpoint of unfolding transition, higher the Cm, more stable is the nature of the protein. A
H20
greater ΔGD value for FgTGR than that of FgTGRsec further validates the more stable nature
of FgTGR. The lesser stability of the full-length enzyme could be explained by the presence of
Sec in the C-terminal that makes the protein flexible to be catalytically more active. This type of
flexibility could be also a prerequisite for facilitating proper binding/positioning of the cofactors
and substrates into their respective pockets, which also correlates with our activity results. The
decrease in the fluorescence polarization as well as the GR and DTNB-based TrxR activity of
FgTGRsec and FgTGR suggests perturbation in the binding of the co-factors to the enzyme. Our
result suggests that slight variations in binding/orientation of the FAD may lead to complete loss
of enzymatic activity of flavoproteins due to the release of protein-bound FAD molecule [66,
6
8]. The comparative unfolding of FgTGRsec and ∆NTD-FgTGRsec showed that the Cm of
NTD-FgTGRsec is less than FgTGRsec, indicating that it is reasonably less stable than
∆
FgTGRsec. This suggests that the presence of the Grx domain enhances the stability of the
enzyme and may be attributed to increased favorable interactions between the domains. These
results demonstrate the role of the Grx domain in maintaining the integrity of the FgTGRsec
structure and that the absence of the Grx domain in the ∆NTD-FgTGRsec makes the structure
susceptible to denaturation at lower GdnHCl concentration.
5
. CONCLUSIONS
TGR plays an indispensible role in the redox biochemistry of parasitic flukes. Several studies
validated TGR as a novel drug target and identified potential leads that show great promise for
treatment of parasitic helminth infections by disrupting redox homeostasis. We investigated the
biochemical and structural properties of different variants of F. gigantica TGR to understand the
role of Sec and the Grx domain. Our results showed that Sec597 is responsible for higher TrxR
and GR activity of FgTGRsec and the N-terminal Grx domain positively regulates the TrxR
activity of FgTGRsec. The unfolding experiments suggested that FgTGR without Sec was more
stable in solution than FgTGRsec and ∆NTD-FgTGRsec. The unique properties of TGR in F.
gigantica make it a promising target for developing new treatments for fascioliasis.
1
8

ACCEPTED MANUSCRIPT
Acknowledgements
The authors thank Dr. Rajanish Giri, IIT Mandi for help with CD experiments. PK thanks NEHU
for providing Non-NET fellowship. RS and HS thank UGC, New Delhi, India for providing
fellowships.
Competing interest
The authors declare that there are no competing interests.
Author’s contributions
Conceived and designed the experiments: PK, RS, HS, TT.
Performed the experiments: PK, RS.
Analyzed the data: PK, RS, HS, TT.
Contributed reagents/materials/analysis tools: PK, RS, HS, TT.
Wrote the paper: PK, RS, HS, TT.
Abbreviations
Thioredoxin glutathione reductase, TGR; glutaredoxin, Grx; thioredoxin reductase, TrxR;
glutathione reductase, GR; Sec containing TGR, FgTGRsec; TGR without Sec, FgTGR; TGRsec
without the N-terminal Grx domain, ∆NTD-FgTGRsec; auranofin, AF; selenocysteine, Sec; 5,5′-
diothiobis(2-nitrobenzoic acid), DTNB; oxidized glutathione, GSSG; reduced glutathione, GSH;
triethylphosphine-gold, TP gold; midpoint of unfolding transition, Cm; free energy of
H20
stabilization in the absence of denaturants, ΔGD
.
REFERENCES
[
1] J. Nordberg, E.S. Arner, Reactive oxygen species, antioxidants, and the mammalian
thioredoxin system, Free Radic Biol Med, 31 (2001) 1287-1312.
2] C. Berndt, C.H. Lillig, A. Holmgren, Thioredoxins and glutaredoxins as facilitators of protein
folding, Biochim Biophys Acta, 1783 (2008) 641-650.
3] T. Tripathi, S. Suttiprapa, B. Sripa, Unusual thiol-based redox metabolism of parasitic flukes,
Parasitol Int, 66 (2017) 390-395.
[
[
1
9

ACCEPTED MANUSCRIPT
[
4] G. Salinas, M.E. Selkirk, C. Chalar, R.M. Maizels, C. Fernandez, Linked thioredoxin-
glutathione systems in platyhelminths, Trends Parasitol, 20 (2004) 340-346.
5] M. Bonilla, S.M. Denicola A Fau - Marino, V.N. Marino Sm Fau - Gladyshev, G. Gladyshev
[
Vn Fau - Salinas, G. Salinas, Linked thioredoxin-glutathione systems in platyhelminth parasites:
alternative pathways for glutathione reduction and deglutathionylation, J. Biol. Chem., 286(7)
(2011) 4959-4967.
[
6] L. Otero, M. Bonilla, A.V. Protasio, C. Fernandez, V.N. Gladyshev, G. Salinas, Thioredoxin
and glutathione systems differ in parasitic and free-living platyhelminths, BMC Genomics, 11
(2010) 237.
[
7] A.N. Kuntz, E. Davioud-Charvet, A.A. Sayed, L.L. Califf, J. Dessolin, E.S. Arner, D.L.
Williams, Thioredoxin glutathione reductase from Schistosoma mansoni: an essential parasite
enzyme and a key drug target, PLoS Med, 4 (2007) e206.
[
8] D.L. Williams, M. Bonilla, V.N. Gladyshev, G. Salinas, Thioredoxin glutathione reductase-
dependent redox networks in platyhelminth parasites, Antioxidants & redox signaling, 19 (2013)
35-745.
9] G.M. Mkoji, J.M. Smith, R.K. Prichard, Antioxidant systems in Schistosoma mansoni:
7
[
Evidence for their role in protection of the adult worms against oxidant killing, International
Journal for Parasitology, 18 (1988) 667-673.
[
[
10] J. Lu, A. Holmgren, Selenoproteins, J Biol Chem, 284 (2009) 723-727.
11] D.L. Hatfield, M.J. Berry, V.N. Gladyshev, Selenium: its molecular biology and role in
human health, Springer Science & Business Media, 2011.
12] Y. Li, Q. Wu, Y. Peng, F. Huang, X. Li, L. Chen, D. Shi, X. Zhou, X. Fan, Expression,
[
crystallization and preliminary X-ray diffraction analysis of thioredoxin glutathione reductase
from Schistosoma japonicum in complex with FAD, Acta Crystallogr F Struct Biol Commun, 70
(2014) 92-96.
[
13] J.L. Rendon, I.P. del Arenal, A. Guevara-Flores, A. Uribe, A. Plancarte, G. Mendoza-
Hernandez, Purification, characterization and kinetic properties of the multifunctional
thioredoxin-glutathione reductase from Taenia crassiceps metacestode (cysticerci), Mol Biochem
Parasitol, 133 (2004) 61-69.
2
0

ACCEPTED MANUSCRIPT
[
14] H.-H. Huang, L. Day, C.L. Cass, D.P. Ballou, C.H. Williams, D.L. Williams, Investigations
on the Catalytic Mechanism of Thioredoxin Glutathione Reductase (TGR) from Schistosoma
mansoni, Biochemistry, 50 (2011) 5870-5882.
[
15] F. Angelucci, D. Dimastrogiovanni, G. Boumis, M. Brunori, A.E. Miele, F. Saccoccia, A.
Bellelli, Mapping the catalytic cycle of Schistosoma mansoni thioredoxin glutathione reductase
by X-ray crystallography, J Biol Chem, 285 (2010) 32557-32567.
[16] A. Guevara-Flores, J.P. Pardo, J.L. Rendon, Hysteresis in thioredoxin-glutathione reductase
(TGR) from the adult stage of the liver fluke Fasciola hepatica, Parasitol Int, 60 (2011) 156-160.
[17] M. Bonilla, A. Denicola, S.V. Novoselov, A.A. Turanov, A. Protasio, D. Izmendi, V.N.
Gladyshev, G. Salinas, Platyhelminth mitochondrial and cytosolic redox homeostasis is
controlled by a single thioredoxin glutathione reductase and dependent on selenium and
glutathione, J Biol Chem, 283 (2008) 17898-17907.
[
18] C. Brandstaedter, K. Fritz-Wolf, S. Weder, M. Fischer, B. Hecker, S. Rahlfs, K. Becker,
Kinetic characterization of wild-type and mutant human thioredoxin glutathione reductase
defines its reaction and regulatory mechanisms, The FEBS journal, (2017).
[
[
19] WHO, WHO estimates of the global burden of foodborne diseases, in, 2015.
20] S. Mas-Coma, M.A. Valero, M.D. Bargues, Fascioliasis, Adv Exp Med Biol, 766 (2014) 77-
1
14.
[
21] S. Mas-Coma, R. Angles, J.G. Esteban, M.D. Bargues, P. Buchon, M. Franken, W. Strauss,
The Northern Bolivian Altiplano: a region highly endemic for human fascioliasis, Trop Med Int
Health, 4 (1999) 454-467.
[
22] M. Parkinson, S.M. O'Neill, J.P. Dalton, Endemic human fasciolosis in the Bolivian
Altiplano, Epidemiol Infect, 135 (2007) 669-674.
[
[
23] G.C. Coles, Anthelmintic activity of triclabendazole, J Helminthol, 60 (1986) 210-212.
24] G.B. Mitchell, L. Maris, M.A. Bonniwell, Triclabendazole-resistant liver fluke in Scottish
sheep, Vet Rec, 143 (1998) 399.
25] G.P. Brennan, I. Fairweather, A. Trudgett, E. Hoey, McCoy, M. McConville, M. Meaney,
[
M. Robinson, N. McFerran, L. Ryan, C. Lanusse, L. Mottier, L. Alvarez, H. Solana, G. Virkel,
P.M. Brophy, Understanding triclabendazole resistance, Exp Mol Pathol, 82 (2007) 104-109.
2
1

ACCEPTED MANUSCRIPT
[
26] Y.M. Brockwell, T.P. Elliott, G.R. Anderson, R. Stanton, T.W. Spithill, N.C. Sangster,
Confirmation of Fasciola hepatica resistant to triclabendazole in naturally infected Australian
beef and dairy cattle, Int J Parasitol Drugs Drug Resist, 4 (2013) 48-54.
[
27] D. Gordon, R. Zadoks, P. Skuce, N. Sargison, Confirmation of triclabendazole resistance in
liver fluke in the UK, Vet Rec, 171 (2012) 159-160.
28] J.M. Kelley, T.P. Elliott, T. Beddoe, G. Anderson, P. Skuce, T.W. Spithill, Current Threat
of Triclabendazole Resistance in Fasciola hepatica, Trends Parasitol, 32 (2016) 458-469.
29] L. Song, J. Li, S. Xie, C. Qian, J. Wang, W. Zhang, X. Yin, Z. Hua, C. Yu, Thioredoxin
glutathione reductase as a novel drug target: evidence from Schistosoma japonicum, PLoS One,
(2012) e31456.
30] A. Caroli, S. Simeoni, R. Lepore, A. Tramontano, A. Via, Investigation of a potential
[
[
7
[
mechanism for the inhibition of SmTGR by Auranofin and its implications for Plasmodium
falciparum inhibition, Biochem Biophys Res Commun, 417 (2012) 576-581.
[
31] R. Shukla, H. Shukla, P. Kalita, T. Tripathi, Structural insights into natural compounds as
inhibitors of Fasciola gigantica thioredoxin glutathione reductase, J Cell Biochem, (2017).
32] B.J. Neves, R.F. Dantas, M.R. Senger, C.C. Melo-Filho, W.C. Valente, A.C. de Almeida,
[
J.M. Rezende-Neto, E.F. Lima, R. Paveley, N. Furnham, E. Muratov, L. Kamentsky, A.E.
Carpenter, R.C. Braga, F.P. Silva-Junior, C.H. Andrade, Discovery of New Anti-Schistosomal
Hits by Integration of QSAR-Based Virtual Screening and High Content Screening, J Med
Chem, 59 (2016) 7075-7088.
[
33] T. Li, P.D. Ziniel, P.Q. He, V.P. Kommer, G.J. Crowther, M. He, Q. Liu, W.C. Van
Voorhis, D.L. Williams, M.W. Wang, High-throughput screening against thioredoxin glutathione
reductase identifies novel inhibitors with potential therapeutic value for schistosomiasis, Infect
Dis Poverty, 4 (2015) 40.
[
34] F. Ross, P. Hernandez, W. Porcal, G.V. Lopez, H. Cerecetto, M. Gonzalez, T. Basika, C.
Carmona, M. Flo, G. Maggioli, M. Bonilla, V.N. Gladyshev, M. Boiani, G. Salinas,
Identification of thioredoxin glutathione reductase inhibitors that kill cestode and trematode
parasites, PLoS One, 7 (2012) e35033.
[
35] C.C. Melo-Filho, R.F. Dantas, R.C. Braga, B.J. Neves, M.R. Senger, W.C. Valente, J.M.
Rezende-Neto, W.T. Chaves, E.N. Muratov, R.A. Paveley, N. Furnham, L. Kamentsky, A.E.
2
2

ACCEPTED MANUSCRIPT
Carpenter, F.P. Silva-Junior, C.H. Andrade, QSAR-Driven Discovery of Novel Chemical
Scaffolds Active against Schistosoma mansoni, J Chem Inf Model, 56 (2016) 1357-1372.
[
36] A. Gupta, M. Kesherwani, D. Velmurugan, T. Tripathi, Fasciola gigantica thioredoxin
glutathione reductase: Biochemical properties and structural modeling, Int J Biol Macromol, 89
(2016) 152-160.
[
37] C.N. Pace, Determination and analysis of urea and guanidine hydrochloride denaturation
curves, Methods Enzymol, 131 (1986) 266-280.
38] H. Shukla, R. Shukla, A. Sonkar, T. Pandey, T. Tripathi, Distant Phe345 mutation
[
compromises the stability and activity of Mycobacterium tuberculosis isocitrate lyase by
modulating its structural flexibility, Sci Rep, 7 (2017) 1058.
[
39] T. Tripathi, S. Rahlfs, K. Becker, V. Bhakuni, Structural and stability characteristics of a
monothiol glutaredoxin: glutaredoxin-like protein 1 from Plasmodium falciparum, Biochim
Biophys Acta, 1784 (2008) 946-952.
[
40] T. Tripathi, Calculation of thermodynamic parameters of protein unfolding using far-
ultraviolet circular dichroism, J Protein Proteomics, 4 (2013) 85-91.
41] T. Tripathi, A. Roseler, S. Rahlfs, K. Becker, V. Bhakuni, Conformational stability and
energetics of Plasmodium falciparum glutaredoxin, Biochimie, 92 (2010) 284-291.
42] A. Fiser, A. Sali, Modeller: generation and refinement of homology-based protein structure
models, Methods Enzymol, 374 (2003) 461-491.
43] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng, T.E.
[
[
[
Ferrin, UCSF Chimera--a visualization system for exploratory research and analysis, J Comput
Chem, 25 (2004) 1605-1612.
[
44] M. Wiederstein, M.J. Sippl, ProSA-web: interactive web service for the recognition of
errors in three-dimensional structures of proteins, Nucleic Acids Res, 35 (2007) W407-410.
45] R.A. Laskowski, MacArthur, M.W., Moss, D.S., and Thornton, J.M., PROCHECK: a
[
program to check the stereochemical quality of protein structures., Journal of applied
crystallography 26 (1993) 283-291.
[
46] R. Luthy, J.U. Bowie, D. Eisenberg, Assessment of protein models with three-dimensional
profiles, Nature, 356 (1992) 83-85.
47] R. Shukla, P.B. Chetri, A. Sonkar, M.Y. Pakharukova, V.A. Mordvinov, T. Tripathi,
Identification of novel natural inhibitors of Opisthorchis felineus cytochrome P450 using
[
2
3

ACCEPTED MANUSCRIPT
structure-based screening and molecular dynamic simulation, J Biomol Struct Dyn, (2017) 1-
1
03.
[
48] R. Shukla, H. Shukla, P. Kalita, A. Sonkar, T. Pandey, D.B. Singh, A. Kumar, T. Tripathi,
Identification of potential inhibitors of Fasciola gigantica thioredoxin1: computational screening,
molecular dynamics simulation, and binding free energy studies, J Biomol Struct Dyn, (2017) 1-
1
6.
49] R. Thomsen, M.H. Christensen, MolDock: a new technique for high-accuracy molecular
docking, J Med Chem, 49 (2006) 3315-3321.
50] J.T. Vivian, P.R. Callis, Mechanisms of tryptophan fluorescence shifts in proteins, Biophys
J, 80 (2001) 2093-2109.
[
[
[
[
51] J.R. Lakowicz, Principles of Fluoresence Spectroscopy, Springer US, 1983.
52] A. Agorio, C. Chalar, S. Cardozo, G. Salinas, Alternative mRNAs arising from trans-
splicing code for mitochondrial and cytosolic variants of Echinococcus granulosus thioredoxin
Glutathione reductase, J Biol Chem, 278 (2003) 12920-12928.
[
53] H.M. Alger, D.L. Williams, The disulfide redox system of Schistosoma mansoni and the
importance of a multifunctional enzyme, thioredoxin glutathione reductase, Mol Biochem
Parasitol, 121 (2002) 129-139.
[
54] D. Su, S.V. Novoselov, Q.A. Sun, M.E. Moustafa, Y. Zhou, R. Oko, D.L. Hatfield, V.N.
Gladyshev, Mammalian selenoprotein thioredoxin-glutathione reductase. Roles in disulfide bond
formation and sperm maturation, J Biol Chem, 280 (2005) 26491-26498.
[
55] F. Angelucci, A.E. Miele, G. Boumis, D. Dimastrogiovanni, M. Brunori, A. Bellelli,
Glutathione reductase and thioredoxin reductase at the crossroad: the structure of Schistosoma
mansoni thioredoxin glutathione reductase, Proteins, 72 (2008) 936-945.
[
56] F. Angelucci, A.A. Sayed, D.L. Williams, G. Boumis, M. Brunori, D. Dimastrogiovanni,
A.E. Miele, F. Pauly, A. Bellelli, Inhibition of Schistosoma mansoni thioredoxin-glutathione
reductase by auranofin: structural and kinetic aspects, J Biol Chem, 284 (2009) 28977-28985.
[
57] G. Salinas, W. Gao, Y. Wang, M. Bonilla, L. Yu, A. Novikov, V.G. Virginio, H.B. Ferreira,
M. Vieites, V.N. Gladyshev, D. Gambino, S. Dai, The Enzymatic and Structural Basis for
Inhibition of Echinococcus granulosus Thioredoxin Glutathione Reductase by Gold(I),
Antioxidants & redox signaling, 27 (2017) 1491-1504.
2
4

ACCEPTED MANUSCRIPT
[
58] L.J. Song, C.X. Yu, S.Y. Xie, X.R. Yin, C.Y. Qian, J. Wang, W. Zhang, X.D. Ke,
Expression and characterization of thioredoxin glutathione reductase of Schistosoma japonicum,
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi, 23 (2011) 412-418.
[
59] N. Changklungmoa, P. Kueakhai, K. Sangpairoj, P. Chaichanasak, W. Jaikua, S.
Riengrojpitak, P. Sobhon, K. Chaithirayanon, Molecular cloning and characterization of Fasciola
gigantica thioredoxin-glutathione reductase, Parasitol Res, 114 (2015) 2119-2127.
[
60] M.V. Gerashchenko, D. Su, V.N. Gladyshev, CUG start codon generates
thioredoxin/glutathione reductase isoforms in mouse testes, J Biol Chem, 285 (2010) 4595-4602.
61] D. Steinmann, T. Nauser, W.H. Koppenol, Selenium and sulfur in exchange reactions: a
comparative study, The Journal of organic chemistry, 75 (2010) 6696-6699.
62] K.P. Bhabak, B.J. Bhuyan, G. Mugesh, Bioinorganic and medicinal chemistry: aspects of
gold (I)-protein complexes, Dalton Transactions, 40 (2011) 2099-2111.
63] S.S. Gunatilleke, C.A. de Oliveira, J.A. McCammon, A.M. Barrios, Inhibition of cathepsin
B by Au(I) complexes: a kinetic and computational study, J Biol Inorg Chem, 13 (2008) 555-
61.
64] T.W. Gilberger, R.D. Walter, S. Muller, Identification and characterization of the functional
[
[
[
5
[
amino acids at the active site of the large thioredoxin reductase from Plasmodium falciparum, J
Biol Chem, 272 (1997) 29584-29589.
[
65] W. Brandt, L.A. Wessjohann, The functional role of selenocysteine (Sec) in the catalysis
mechanism of large thioredoxin reductases: proposition of a swapping catalytic triad including a
Sec-His-Glu state, Chembiochem, 6 (2005) 386-394.
[
66] A. De la Rosa Miguel, J. Márquez Antonio, M. Vega José, Dissociation of FAD from the
NAD(P)H-Nitrate Reductase Complex from Ankistrodesmus braunii and Role of Flavin in
Catalysis, Zeitschrift für Naturforschung C, 37 (2014) 24-30.
[
67] J. Zou, P. Taylor, J. Dornan, S.P. Robinson, M.D. Walkinshaw, P.J. Sadler, First Crystal
Structure of a Medicinally Relevant Gold Protein Complex: Unexpected Binding of, Angew
Chem Int Ed Engl, 39 (2000) 2931-2934.
[
68] H.Y. Neujahr, Effect of anions, chaotropes, and phenol on the attachment of flavin adenine
dinucleotide to phenol hydroxylase, Biochemistry, 22 (1983) 580-584.
2
5

ACCEPTED MANUSCRIPT
Figure Legends
Figure 1. Purification of the recombinant proteins over Ni-NTA agarose. (A) SDS-PAGE
analysis of the purified proteins. Lanes 1–4 represent molecular weight markers, purified
FgTGRsec, FgTGR and ∆NTD-FgTGRsec, respectively. (B) SEC profile of native FgTGRsec,
FgTGR and ∆NTD-FgTGRsec. Molecular weight and oligomeric status of the recombinant
proteins were determined on Superdex™ 200 10/300 GL column at pH 8.0 and 25 °C. The
column was calibrated with standard molecular weight markers: Aldolase (158kDa), conalbumin
(75 kDa), ovalbumin (44kDa) and ribonuclease A (13.7 kDa).
Figure 2. Structural features of recombinant proteins. (A) Tryptophan emission fluorescence
spectra. (B) Far-UV CD spectra. In these figures, the black, red and green lines represent the data
for FgTGRsec, FgTGR and ∆NTD-FgTGRsec samples, respectively.
Figure 3. Comparison of binding affinity of DTNB and GSSG with FgTGRsec and FgTGR
using fluorescence quenching and activity assays. (A) Stern–Volmer plot for the fluorescence
intensity (F /F ) of FgTGRsec and FgTGR upon DTNB binding. (B) Comparative TrxR activity
0
1
of FgTGRsec and FgTGR with DTNB. (C) Stern–Volmer plot for the fluorescence intensity
F /F ) of FgTGRsec and FgTGR upon GSSG binding. (D) Comparative GR activity of
(
0
1
FgTGRsec and FgTGR with GSSG. In these figures the solid squares with solid line represents
FgTGRsec and the hollow squares with dotted line represents FgTGR, respectively.
Figure 4. Comparison of DTNB binding affinity to FgTGRsec and ∆NTD-FgTGRsec with
fluorescence quenching and activity assays. (A) Stern–Volmer plot for the fluorescence
intensity (F /F ) of ∆NTD-FgTGRsec upon DTNB binding. (B) Comparative TrxR activity of
0
1
FgTGRsec and ∆NTD-FgTGRsec with DTNB. In these figures the solid squares with solid line
represents FgTGRsec and the hollow circles with dotted line represents ∆NTD-FgTGRsec,
respectively.
Figure 5. Inhibition of FgTGRsec by AF. (A) Stern–Volmer plot for the fluorescence intensity
(
F /F ) of FgTGRsec upon AF binding, where the solid squares represents FgTGRsec (B)
0 1
2
6

ACCEPTED MANUSCRIPT
Inhibition of TrxR activity and GR activity of FgTGRsec by AF. (C) Calculation of IC50 by
plotting percent inhibition of activity against log of [AF]. In figures (B) and (C) The solid
squares represents Trx activity and hollow squares represents GR activity of FgTGRsec,
respectively.
Figure 6. The potential binding poses for the Auranofin and TP-gold moiety. (A) Auranofin,
and, (B) TP-gold moiety. The sphere represents the gold atom. Red dotted lines represent the
hydrogen bonds.
Figure 7. Comparison of GdnHCl induced unfolding of FgTGRsec and FgTGR. (A) Effect
of increasing GdnHCl concentrations on the CD ellipticity at 222 nm and the tryptophan
emission wavelength maxima of FgTGRsec and FgTGR. The data is presented as fraction
unfolded with the value observed in absence of GdnHCl taken as 0%. (B) Calculation of free
H2O
energy, ∆GD
of unfolding in absence of denaturants from both CD and fluorescence signals.
In figures (A) and (B) the solid squares, solid circles represent data for fluorescence and CD of
FgTGRsec and; the hollow squares and hollow circles represent data for FgTGR, respectively.
(
C) and (D) Effect of GdnHCl on activity and polarization of FAD. The solid circles (black) and
solid circles (red) represent activity of FgTGRsec and FgTGR respectively. The solid triangles
black) and solid triangles (red) represent change in FAD polarization of FgTGRsec and FgTGR,
(
respectively.
Figure 8. Comparison of GdnHCl induced unfolding of FgTGRsec and ∆NTD-FgTGRsec.
(A) Effect of increasing GdnHCl concentrations on the CD ellipticity at 222 nm and the
tryptophan emission wavelength maxima of FgTGRsec and ∆NTD-FgTGRsec. The data is
presented as fraction unfolded with the value observed in absence of GdnHCl taken as 0 %. In
figures (A) and (B) the solid squares, solid circles represent data for fluorescence and CD of
FgTGRsec and; the hollow squares and hollow circles represent data for ∆NTD-FgTGRsec,
H2O
respectively. (B) Calculation of free energy, ∆G
of unfolding in absence of denaturants from
both CD and fluorescence signals. (C) Effect of GdnHCl on TrxR activity and polarization of
FAD. The solid circles (black) and solid circles (red) represent TrxR activity of FgTGRsec and
2
7

ACCEPTED MANUSCRIPT
∆NTD-FgTGRsec respectively. The solid triangles (black) and solid triangles (red) represent
change in FAD polarization of FgTGRsec and ∆NTD-FgTGRsec, respectively.
2
8

ACCEPTED MANUSCRIPT
-1
Table 1. Stern Volmer quenching constants (K ) in µM .
SV
Quencher
DTNB
FgTGRsec
FgTGR
∆NTD-FgTGRsec
-3
-3
-3
60.6 x 10
42.5 x 10
52.2 x 10
-
3
-3
GSSG
0.7 x 10
0.5 x 10
-
ND
-
-3
Auranofin
58.8 x 10
2
9