Computer-aided Molecular Design To Improve The Thermotability Of Glutamate Decarboxylase

Glutamate decarboxylase?GAD;EC 4.1.1.15?is a highly effcient enzyme that specifically catalyzes the decarboxylation of L-glutamic acid to produce?-aminobutyric acid?GABA?in the presence of pyridoxal-5-phosphate?PLP?,which has important application value in the fields of food,medicine and chemical industry.However,glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry.Improving their thermostability is a critical problem to be addressed in industrial production.In this study,the glutamate decarboxylase derived from L.brevis CGMCC 1306was used as model.The strategies of homologous thermophilic enzyme sequence alignment and increase of disulfide bonds were selected to design point mutations,and mutants with significantly improved thermostability were successfully obtained.The specific research contents are as follows:1.Five GADs with high thermostability and high catalytic activity were retrieved from Brenda enzyme database,then amino acid residues that are conserved within thermophilic proteins but absent in mesophilic proteins were selected by sequence alignment.To estimate the Gibbs energy effect of these substitutions,five sites with lower energy were selected for experimental verification.The stability analysis results showed that the T₅₀¹⁵values of D203E and S325A were 62.6?and 61.7?,which were 2.3?and 1.4?higher than wild-type enzyme?60.3??,respectively.And the k_cat/K_mvalue of S325A is 61.9%higher than the wild type.Although the thermostability of mutant N193D has decreased,its k_cat/K_mvalue is 4.00 s^-1·m M^-1,which is 55.6%higher than that of wild type.Subsequently,three double mutants were obtained by combining the three enzymes in pairs,and the T₅₀¹⁵of the mutant enzymes N193D-D203E and N193D-S325A were 63.2?and 62.1?,respectively,were similar to each single mutant enzyme,and the k_cat/K_mof the two mutants were2.04 s^-1·m M^-1and 2.64 s^-1·m M^-1,respectively.The catalytic efficiency of the two mutants was lower than that of the single mutant enzymes.2.The computer program Db D V2.12 predicts locations where disulfide bonds could be introduced without introducing strain in the folded form.These sites were evaluated by B-factor analysis and energy calculation,and four sites were selected for the construction of mutants.By thermostability analysis,the T₅₀¹⁵values of the mutant191C-291C,369C-379C showed a 3.0?,4.3?increase to the wild-type enzyme,respectively.Furthermore,the optimal temperature of the mutant enzyme 369C-379C was increased to 56?.The kcat/Km values of the mutants 191C-219C and369C-379C were 1.01 s^-1·m M^-1and 2.25 s^-1·m M^-1,which were 0.39 and 0.88 times of the corresponding values of the wild-type enzyme,respectively.Although the thermostability of the two mutants were improved,the enzyme 191C-291C has a serious negative effect on the catalytic activity,while the catalytic efficiency of the enzyme 369C-379C has not been significantly reduced.3.In order to further explain the effect of the mutation site on the conformation of the protein,RMSD and RMSF in the molecular dynamics simulation trajectories of wild and mutant enzymes were calculated to compare the fluctuations of the potential energy for the whole system.Molecular dynamics simulation revealed that the overall average RMSD values of D203E,S325A,191C-219C,369C-379C were lower than that of wild-type protein structure during 10 ns simulation at 300 K.In addition,according to the analysis of the PIC online server,the improved thermotability of the mutant enzymes S325A and 369C-379C were related to the formation of new hydrophobic bonds?325-78,325-82?and disulfide bonds?369-379?.These regions in the wild-type structure is more flexible than in mutants S325A and 369C-379C structures according to RMSF,indicating that more stable conformation was achieved.