Isolation and mechanism analysis of a catalytic efciency improved L‑aspartateβ‑decarboxylase toward 3‑methylaspartic acid

L-Aspartateβ-decarboxylase from Acinetobacter radioresistens(ArASD)has been modifed to convert 3-methylaspartic acid into 2-aminobutyric acid,which activated a novel process for biosynthesis of 2-aminobutyric acid.However,the process is limited by the low activity of the ArASD.Here,the activity of ArASD was signifcantly improved by modifcation based on sequence alignment and structural analysis.The 38th residue of ArASD is speculated to be the key residue for regulating the conformation of the internal aldimine,and site-directed mutagenesis on R38 residue was carried out.A variant,K18A/R38K/V287I,with 2.2 times higher specifc activity was isolated.Molecular dynamics simulation indicated that the torsion angle of the imine bond of the variant decreased,which was benefcial to the protonation of the internal aldimine and the increase in the initial energy of the enzyme.Therefore,the energy barrier of the transition state was reduced,resulting in improved catalytic activity toward 3-methylaspartic acid.These results provide a reference and a new point of view for enzyme modifcation by increasing the energy of the initial state.

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

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.