Background: Glutathione transferases (GSTs, EC. 188.8.131.52) form a large group of multifunctional
enzymes that are involved in the metabolism and inactivation of a wide range of endogenous
and xenobiotic compound as well as in cell regulation and response to several biotic and abiotic
Objectives: In the present work, we report the comparative analysis of the structural and functional
features of two isoenzymes (GmGSTU5-5 and GmGSTU8-8) of the glutathione transferase (GST)
family from Glycine max.
Methods: Full-length cDNA clones of GmGSTU5-5 and GmGSTU8-8 were derived from RT-PCR
of RNA isolated from soybean seedlings and were cloned into a T7 expression vector. Τhe recombinant
enzymes were expressed in E. coli and purified by affinity chromatography. Substrate specificity,
kinetic and inhibition analysis were carried out towards a range of different xenobiotic compounds
and GSH analogues. The thermal stability of the enzymes was also evaluated using activity
assays and differential scanning fluorimetry.
Results: Analysis of substrate specificity using a range of thiol substrates and electrophilic compounds
suggested that both isoenzymes display broad and overlapping specificities. They are capable
of detoxifying major stress-induced toxic products. Study of their ligandin-binding properties
by kinetic analysis and molecular modelling indicated that both GmGSTU5-5 and GmGSTU8-8
bind a range of secondary metabolites and plant hormones, suggesting a role in transport or storage
of bioactive compounds. Thermostability analysis showed that GmGSTU5-5 and GmGSTU8-8
display extraordinary thermal stability, compared to other plant GSTs.
Conclusion: Our results suggest that GmGSTU5-5 and GmGSTU8-8 display different or overlapping
substrate specificities and kinetic properties. The biological role of GmGSTU5-5 and
GmGSTU8-8 may be relevant to the detoxification of toxic compounds or the binding of bioactive
metabolites that function in cell regulation and stress defence mechanisms.