Study On Key Sites Of Glutamate Decarboxylase Active Center Of Lactobacillus Brevis

Glutamate decarboxylase?EC 4.1.1.15,GAD?is a pyridoxal 5-phosphate?PLP?dependent enzyme,which catalyses the irreversible?-decarboxylation of L-glutamic acid to?-aminobutyric acid?GABA?and CO₂.As a major inhibitory neurotransmitter present in the vertebrate central nervous system,GABA possesses various important biological properties,such as diuretic,anti-hypertension,anti-oxidant,anti-fatigue,tranquilizing,sleep improvement,stimulation of immune cells,treatment for chronic alcohol-related symptoms and anti-diabetic effects.As a consequence,GABA is used considerably in pharmaceuticals and functional foods.In our previous work,the crystal structure of GAD from Lactobacillus brevis CGMCC 1306 has been successfully solved.To gain better insights into its catalytic mechanism and structure-function relationship,the structure-guided site-directed mutagenesis of GAD was conducted for residues around the PLP,substrate binding sites and flexible loop with higher crystallographic B-factors.The results are as follows:?1?The interactions between target protein GAD and PLP were observed and analyzed using Discovery Studio 2018 software.The binding sites were defined within 10?distance around PLP according to the crystal structures of GAD.The results showed that the changes in catalytic efficiency(k_cat/K_m)of C168A(0.171?mmol/L?^-1?s^-1)was a decrease by a factor of 0.235.The substrate-binding affinity?K_m?changes?56.894±4.568 mmol/L?were an increase by a factor of 1.38,indicating a possible decrease in substrate affinity.The residues which are involved in interactions with PLP in GAD,play important roles in supporting the catalytic activity of the PLP cofactor.These amino acid residues contribute to stabilizing the phosphate group and pyridine ring of PLP cofactor,and anchoring it inside the active site.?2?To further elaborate the atomic details of active substrate-GAD interactions,the L-Glu was docked with GAD/PLP using CDOCKER module of Discovery studio 2018.All calculations for protein-fixed ligand-flexible docking were done.Of all the mutants,the K_m?39.447±2.652 mmol/L?and the k_cat/K_m(1.227?mmol/L?^-1?s^-1)of the mutant enzyme T215A were 0.956 times?41.283±2.279 mmol/L?and 1.587 times(0.773?mmol/L?^-1?s^-1)of the wild enzyme,respectively.To further investigate the role of T215 in GAD,semi-saturation mutagenesis of T215 was performed.T215A was the only mutant with substantially increased activity,while the other mutants of T215 retained extremely low decarboxylase activity?<5%?suggesting that reduced steric hindrance at position 215 is advantageous for improving enzyme activity.And the higher hydrophobicity at position 215 might contribute to a-decarboxylation of L-Glu.?3?The loop with the higher B-factor value is located on the protein surface and proximal to the active site of the adjacent monomer using PyMOL,enabling residues in the loop region to participate in forming the entrance to the active site.The mutants were obtained by deleting the amino acid residues of a dynamic surface loop Y308-E312 by site-directed mutagenesis.The results showed that the mutant enzymes?Y308del,?YL308del,?YLG308del,?YLGG308del and?YLGGE308del had no detectable activity in the presence of excess substrate L-Glu.On the contrary,the catalytic activities of the mutant enzymes?L309del,?LG309del,?LGG309del and?LGGE309del were all below 5%of the wild enzyme.It indicated that the reduction in the length of the flexible ring including L309del affects the overall activity of the GAD and is not completely inactivated.Thus,deletion of amino acid residues in the flexible loop?Y308-E312?may disrupt intramolecular interactions in the active center of GAD,making the loop region with higher B-factor value unstable and affecting GAD catalytic activity.In this study,the combination of bioinformatics and protein engineering methods was used to clarify the role of key amino acid residue sites in the GAD active center and its neighbouring loop region,providing a theoretical basis for improving its catalytic efficiency.