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 modeling, 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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The dimeric reaction centre light-harvesting 1 (RC-LH1) core complex of Rhodobacter sphaeroides converts absorbed light energy to a charge separation, and then it reduces a quinone electron and proton acceptor to a quinol. In this issue, Qian and colleagues (pp. 3923–3937) investigate the dimerisation interface between two RC-LH1 monomers, and determine the cryogenic electron microscopy structure of the dimeric complex at 2.9 Å resolution. The cover image shows the role of PufX in imposing a bent conformation on the RC-LH1 dimer complex. The top view is of the cytoplasmic side of the membrane and the bottom view is in the plane of the membrane. Image courtesy of C. Neil Hunter.
A conserved NAG motif is critical to the catalytic activity of galactinol synthase, a key regulatory enzyme of RFO biosynthesis
Prafull Salvi, Bhavnesh Kumar, Nitin Uttam Kamble, Abhijit Hazra, Manoj Majee; A conserved NAG motif is critical to the catalytic activity of galactinol synthase, a key regulatory enzyme of RFO biosynthesis. Biochem J 12 November 2021; 478 (21): 3939–3955. doi: https://doi.org/10.1042/BCJ20210703
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