Enhancing Catalytic Property Of Glutamate Decarboxylase:an Insight From Protein Ramachandran Plot

Glutamate decarboxylase (GAD, EC4.1.1.15) can catalyze the decarboxylation of glutamate into γ-aminobutyrate (GABA) and is the only enzyme of GABA biosynthesis. GABA, a four-carbon non-protein amino acid, has several physiological functions such as hypotensive activity, tranquilizing and allaying excitement, improving sleep quality and enhancing memory. Therefore, GABA has a broad application prospect in food, pharmaceutical, animal husbandry, agriculture and other fields. However, low catalytic activity and poor thermostability of the natural GAD limited itself in the large-scale application of GABA biosynthesis. Based on the prospects in utilizing Lactobacillus brevis GAD for the biosynthesis of GABA, we employed bioinformatics methods to create a more efficient biocatalyst for GABA preparation, thus breaking through the above mentioned bottleneck. Here, we performed site-directed mutagenesis to modify this enzyme to facilitate its utilization in the biosynthesis of GABA according to the Ramachandran plot information of GAD 1407 three-dimensional structure.First of all, according to the Ramachandran plot information of GAD 1407 three-dimensional structure from Lactobacillus brevis CGMCC No.1306, we identified the unstable site K413 as the mutation target, constructed the mutant GAD by site-directed mutagenesis and measured the thermostability and catalytic activity of wide type and mutant GAD. The results showed that the mutant K413A led to a remarkably slower inactivation rate, and its half-life at 50℃ reached 105 min which was 2.1-fold higher than the wild type GAD1407. Moreover, the mutant K413I exhibited 1.6-fold higher activity in comparison with the wide type GAD 1407, but it had no remarkable improvement in thermostability of GAD. The enzymatic properties of the mutant K413A and mutant K413I showed that the mutant enzyme had little difference with wide type on the substrate affinity, but turnover number (kcat) of mutant K413A and mutant K413I increased by 33% and 67%, respectively. Through the analysis of three-dimensional structures of GAD1407 using Swiss Model homology program, we found that the mutant improved catalytic activity or thermostability mainly because of the increase in the hydrophobic interaction among amino acid residues.Then, we explored the unfolding processing of wide type and mutant K413A by fluorescence spectra in different concentrations of guanidine hydrochloride (GdmHCl) or urea. When the excitation wavelength was 280 nm, ultizing GdmHCl as denaturant, the tryptophan largest emission peak of both two enzymes redshifted, tryptophan fluorescence intensity increased at first and then gradually declined; ultizing urea as denaturant, the tryptophan largest emission peak of both two enzymes also redshifted, and tryptophan fluorescence intensity declined. With the increasing concentration of denaturant, the phase diagram of fluorescence showed that it existed a partially folded intermediate no matter the mutant K413A or the wide type, which followed a three-state model. In addition, we also explored the influence of denaturant on catalytic activity of the wide type and mutant K413A. Wide type and mutant K413A reached their half inactivation respectively, when the GdmHCl concentration was about 0.4 mol/L and 0.7 mol/L. Wide type and mutant K413A reached their half inactivation respectively, when the urea concentration was about 1 mol/L and 2 mol/L. Therefore, the mutant K413A showed a more stable performance than wide type under the presence of denaturant.This study not only obtained the mutant GAD with higher catalytic activity or higher thermalstability which applied for GABA biolosythesis, but also set up a methods for predicting effective mutation based on Ramachandran plot information, and providing a new approach to increase GAD thermostability and catalytic activity through rational design.