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    A conserved NAG motif is critical to the catalytic activity of galactinol synthase, a key regulatory enzyme of RFO biosynthesis
    (Portland Press, 2021) Salvi, Prafull; Kumar, Bhavnesh; Kamble, Nitin Uttam; Hazra, Abhijit; Majee, Manoj
    Galactinol synthase (GolS) catalyzes the key regulatory step in the biosynthesis of Raffinose Family Oligosaccharides (RFOs). Even though the physiological role and regulation of this enzyme has been well studied, little is known about active site amino acids and the structure-function relationship with substrates of this enzyme. In the present study, we investigate the active site amino acid and structure function relationship for this enzyme. Using a combination of three-dimensional homology modelling, molecular docking along with a series of deletion, site directed mutagenesis followed by in vitro biochemical and in vivo functional analysis; we have studied active site amino acids and their interaction with the substrate of chickpea and Arabidopsis GolS enzyme. Our study reveals that the GolS protein possesses GT8 family-specific several conserved motifs in which NAG motif plays a crucial role in substrate binding and catalytic activity of this enzyme. Deletion of entire NAG motif or deletion or the substitution (with alanine) of any residues of this motif results in complete loss of catalytic activity in in vitro condition. Furthermore, disruption of NAG motif of CaGolS1 enzyme disrupts it’s in vivo cellular function in yeast as well as in planta. Together, our study offers a new insight into the active site amino acids and their substrate interaction for the catalytic activity of GolS enzyme. We demonstrate that NAG motif plays a vital role in substrate binding for the catalytic activity of galactinol synthase that affects overall RFO synthesis.
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    Stress inducible galactinol synthase of chickpea (CaGolS) implicates in heat and oxidative stress tolerance through reducing stress induced excessive reactive oxygen species accumulation
    (Oxford University Press, 2018) Salvi, Prafull; Kamble, Nitin Uttam; Majee, Manoj
    Raffinose Family Oligosaccharides (RFOs) participates in various aspects of plant physiology and galactinol synthase (GolS, EC 2.4.1.123) catalyzes the key step of RFO biosynthesis. Stress induced accumulation of RFOs particularly galactinol and raffinose has been reported in few plants; however their precise role and mechanistic insight in stress adaptation remain elusive. In present study, we have shown that the GolS activity as well as galactinol and raffinose content are significantly increased in response to various abiotic stresses in chickpea. Transcriptional analysis indicated that the CaGolS1 and CaGolS2 genes are induced in response to different abiotic stresses. Interestingly, heat and oxidative stress preferentially induce CaGolS1 over CaGolS2. Insilco analysis revealed several common yet distinct cis-acting regulatory elements in their 5' upstream regulatory sequences. Further, in vitro biochemical analysis revealed that CaGolS1 enzyme functions better in stressful conditions than CaGolS2 enzyme. Finally, Arabidopsis transgenic plants constitutively overexpressing CaGolS1 or CaGolS2 not only exhibits significantly increased galactinol but also raffinose content and display better growth responses than wild type or vector control plants when exposed to heat and oxidative stress. Further, improved tolerance of transgenic lines are associated with reduced accumulation of reactive oxygen species (ROS) and consequent lipid peroxidation as compared to control plants.Collectively, our data implies that GolS enzyme activity and consequent galactinol and raffinose content are significantly increased in response to stresses to mitigate stress induced growth inhibition by restricting excessive ROS accumulation and consequent lipid peroxidation in plants.