Identification of key residues for the activity of aspartate 4‑decarboxylase towards l‑3‑methylaspartate  

作　　者：Mingzhu Hao Laichuang Han Zhemin Zhou Zhongmei Liu 

机构地区：[1]School of Biotechnology,Jiangnan University,Wuxi,Jiangsu,China

出　　处：《Systems Microbiology and Biomanufacturing》2023年第3期449-456,共8页系统微生物学与生物制造（英文）

基　　金：the National Natural Science Foundation of China(21878125).

摘　　要：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.

关 键 词：Aspartate 4-decarboxylase l-3-methylasparte Substrate-binging pocket Molecular dynamics simulation 

分 类 号：O64[理学—物理化学]