Parallel Strategy Increases the Thermostability and Activity of Glutamate Decarboxylase.

Zhang Zhang,Zhao Zhao,Huang Huang,Mei Mei,Hu Hu

doi:10.3390/molecules25030690

Abstract

Glutamate decarboxylase (GAD; EC 4.1.1.15) is a unique pyridoxal 5-phosphate (PLP)-dependent enzyme that specifically catalyzes the decarboxylation of L-glutamic acid to produce γ-aminobutyric acid (GABA), which exhibits several well-known physiological functions. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, a parallel strategy comprising sequential analysis and free energy calculation was applied to identify critical amino acid sites affecting thermostability of GAD and select proper mutation contributing to improve structure rigidity of the enzyme. Two mutant enzymes, D203E and S325A, with higher thermostability were obtained, and their semi-inactivation temperature (T5015) values were 2.3 °C and 1.4 °C higher than the corresponding value of the wild-type enzyme (WT), respectively. Moreover, the mutant, S325A, exhibited enhanced activity compared to the wild type, with a 1.67-fold increase. The parallel strategy presented in this work proved to be an efficient tool for the reinforcement of protein thermostability.

Highlights

Glutamate decarboxylase (GAD; EC 4.1.1.15) is a highly efficient enzyme that catalyzes the decarboxylation of L-glutamic acid to produce γ-aminobutyric acid (GABA) in the presence of pyridoxal-5-phosphate (PLP)
Due to the safety and environmental friendliness of lactic acid bacteria (LAB), LAB has been widely used in the fermentation of GABA [7]
GAD is a pyridoxal 5-phosphate (PLP)-dependent decarboxylase, an imine linkage between PLP and the active site lysine (K279) ensuring the PLP is properly oriented at the active site for the reaction with the substrate

Summary

Introduction

Glutamate decarboxylase (GAD; EC 4.1.1.15) is a highly efficient enzyme that catalyzes the decarboxylation of L-glutamic acid to produce γ-aminobutyric acid (GABA) in the presence of pyridoxal-5-phosphate (PLP). GAD was first discovered in mammalian brain tissue in 1951, and Paul Y. Sze reported cellular regulation of GAD which has provided important information for understanding. GABA-ergic neurons and their functions [1,2]. GABA exhibits several well-known physiological functions in humans, such as induction of hypotensive effects, anticonvulsant and anti-depression effects, the promotion of hormone secretion, and protection of the liver and kidney [3,4,5,6]. We have cloned the GAD with high activity from Lactobacillus brevis

Methods

Results

Conclusion