Aspartate 4-decarboxylase(ASD)has been modified to obtain the catalytic ability of the unnatural substrate l-3-methylaspartate.However,the mechanism remains to be clarified.In the present study,the semi-rational modif...Aspartate 4-decarboxylase(ASD)has been modified to obtain the catalytic ability of the unnatural substrate l-3-methylaspartate.However,the mechanism remains to be clarified.In the present study,the semi-rational modification was used to identify key residues of importance for the activity towards l-3-methylasparte.The ASD from Pseudomonas dacunhae 21192(PdASD)was used as a template,which showed better activity than the other two ASDs.Four residues proved to be critical for the activity towards l-3-methylasparte,with three located in the active site and one on the surface.Combinatorial variants were constructed to analyze the role of each mutation.The enzymatic properties of the combined variants were determined and compared.The residue at the 17th position was a member of the substrate entrance gate and contributed to the activity by reducing the steric hindrance.The residue at the 37th position was necessary for activity.Two mutations,I288 and V382,exhibited strong epistatic interactions on the activity of ASD.Structural changes in the active site were analyzed by molecular dynamics simulations,and it is proposed that the increased activity of PdASD variants is related to a suitable binding pocket for the substrate.These results provide new evidence for the mechanism ofβ-decarboxylation,which lays the foundation for enhancing the activity of ASD.展开更多
基金the National Natural Science Foundation of China(21878125).
文摘Aspartate 4-decarboxylase(ASD)has been modified to obtain the catalytic ability of the unnatural substrate l-3-methylaspartate.However,the mechanism remains to be clarified.In the present study,the semi-rational modification was used to identify key residues of importance for the activity towards l-3-methylasparte.The ASD from Pseudomonas dacunhae 21192(PdASD)was used as a template,which showed better activity than the other two ASDs.Four residues proved to be critical for the activity towards l-3-methylasparte,with three located in the active site and one on the surface.Combinatorial variants were constructed to analyze the role of each mutation.The enzymatic properties of the combined variants were determined and compared.The residue at the 17th position was a member of the substrate entrance gate and contributed to the activity by reducing the steric hindrance.The residue at the 37th position was necessary for activity.Two mutations,I288 and V382,exhibited strong epistatic interactions on the activity of ASD.Structural changes in the active site were analyzed by molecular dynamics simulations,and it is proposed that the increased activity of PdASD variants is related to a suitable binding pocket for the substrate.These results provide new evidence for the mechanism ofβ-decarboxylation,which lays the foundation for enhancing the activity of ASD.
