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Structural Biology 2018 & STD AIDS 2018
Journal of Genetics and Molecular Biology
|
Volume 2
S e p t e m b e r 0 3 - 0 4 , 2 0 1 8 | B a n g k o k , T h a i l a n d
allied
academies
STD-AIDS AND INFECTIOUS DISEASES
STRUCTURAL BIOLOGY AND PROTEOMICS
&
International Conference on
International Conference on
Joint Event on
J Genet Mol Biol 2018, Volume 2
BIOCHEMICAL & BIOPHYSICAL CHARACTERIZATION AND
THERMODYNAMIC COMPARISON OF DIFFERENT VARIANTS OF THE
REDOX SELENOZYME THIOREDOXIN GLUTATHIONE REDUCTASE OF
FASCIOLA GIGANTICA
Parismita Kalita
and
Timir Tripathi
North Eastern Hill University, India
T
he 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 (ΔNTD-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, 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 ΔNTD-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.