Effects of Substrate-Binding Site Residues on the Biochemical Properties of a Tau Class Glutathione S-Transferase from Oryza sativa.

Xue Yang,Jie Gao,Zhihai Wu,Jinchi Wei

doi:10.3390/genes11010025

Abstract

Glutathione S-transferases (GSTs)—an especially plant-specific tau class of GSTs—are key enzymes involved in biotic and abiotic stress responses. To improve the stress resistance of crops via the genetic modification of GSTs, we predicted the amino acids present in the GSH binding site (G-site) and hydrophobic substrate-binding site (H-site) of OsGSTU17, a tau class GST in rice. We then examined the enzyme activity, substrate specificity, enzyme kinetics and thermodynamic stability of the mutant enzymes. Our results showed that the hydrogen bonds between Lys42, Val56, Glu68, and Ser69 of the G-site and glutathione were essential for enzyme activity and thermal stability. The hydrophobic side chains of amino acids of the H-site contributed to enzyme activity toward 4-nitrobenzyl chloride but had an inhibitory effect on enzyme activity toward 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Different amino acids of the H-site had different effects on enzyme activity toward a different substrate, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Moreover, Leu112 and Phe162 were found to inhibit the catalytic efficiency of OsGSTU17 to 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, while Pro16, Leu112, and Trp165 contributed to structural stability. The results of this research enhance the understanding of the relationship between the structure and function of tau class GSTs to improve the abiotic stress resistance of crops.

Highlights

Glutathione S-transferases (GSTs; EC. 2.5.1.18), widely distributed in aerobic organisms, are a superfamily of multifunctional proteins that were found to play an important role in phase II detoxification [1,2,3,4]
Mutants were relatively unstable thermodynamically among all hydrophobic substrate-binding site (H-site) mutants, with an inactivation temperature of 55 ◦ C. This was especially true for the N109A mutant, whose activity sharply declined after 40 ◦ C. These results suggest that the amino acids forming the H-site have less of an effect on the structural stability of OsGSTU17 than the GSH binding site (G-site) residues
We speculate that the hydrogen bond formed by Lys42 is more important for the correct combination and recognition of GSH than other residues of G-site in OsGSTU17

Summary

Introduction

Glutathione S-transferases (GSTs; EC. 2.5.1.18), widely distributed in aerobic organisms, are a superfamily of multifunctional proteins that were found to play an important role in phase II detoxification [1,2,3,4]. GSTs conjugate glutathione (GSH) with various electrophilic xenobiotic compounds or endogenous substrates to form soluble nontoxic substances [1]. Plant GSTs exert other catalytic or non-catalytic functions. Genes 2020, 11, 25 reactions, GSH-dependent isomerizations, and GSH-dependent thioltransferase reactions [1,3,5,6]. Their non-catalytic functions are as binding or carrier proteins for transporting phytochemicals between cellular compartments [1,2,5,7,8,9,10,11,12,13,14]. Only GSH-dependent detoxification received significant attention in plant GSTs to increase the herbicide tolerance of crop in the past decades [3]

Methods

Results

Discussion

Conclusion