A conserved NAG motif is critical to the catalytic activity of galactinol synthase, a key regulatory enzyme of RFO biosynthesis.

Prafull Salvi,Abhijit Hazra,Manoj Majee,Nitin Uttam Kamble,Bhavnesh Kumar

doi:10.1042/bcj20210703

Prafull Salvi, Abhijit Hazra + Show 3 more

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https://doi.org/10.1042/bcj20210703

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Abstract

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.

Highlights

Galactinol synthase aka inositol -3-α galactosyl transferase (GolS, EC: 2.4.1.123) is a member of Glycosyltransferase family 8 GT8 family glycosyltransferase in CAZy (Carbohydrate Active enzymes) database and is present only in flowering plants [1,2]
The structural analysis of CaGolS1 typically showed a complex structure with seven α-helices and nine β-sheets forming the central core area of protein surrounded by several varied length polypeptide chains on surface of protein
GolS is restricted to the plant kingdom and belongs to GT8 family [4,16]

Summary

Introduction

Galactinol synthase aka inositol -3-α galactosyl transferase (GolS, EC: 2.4.1.123) is a member of Glycosyltransferase family 8 GT8 family glycosyltransferase in CAZy (Carbohydrate Active enzymes) database and is present only in flowering plants [1,2]. In an attempt to identify specific motif/domain and/or amino acid sequence(s) crucial for the catalytic activity of GolS enzyme, we performed in-depth bioinformatic analysis of CaGolS1 using several approaches followed by making a series of deletion or substitution mutants of the residues identified as potentially important for GolS activity. Biochemical analysis of these mutant enzymes in in vitro demonstrated that NAG motif plays a very important role in the substrate binding and catalysis, where any point mutation in the NAG motif turn-out to be a total loss in GolS enzyme activity. The difference in the mean was considered statistically significant if the α

Results

Discussion

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