| L-aspartateβ-decarboxylase(E.C.4.1.1.12,Asd)is the only amino acid decarboxylase capable ofβ-site decarboxylation in nature and is widely found in prokaryotes and eukaryotes.The group obtained an Acinetobacter radioresistens-derived Asd mutant that can catalyze L-3-methylaspartate by molecular modification in the early stage,and achieved the synthesis of L-2-aminobutyric acid from L-glutamate by tandem fusion of glutamate translocase and A.radioresistens-derived Asd mutant.However,the catalytic efficiency of the Asd mutant for the intermediate product of the pathway,L-3-methylaspartate,is still low and it is the rate-limiting enzyme of the pathway;moreover,the mechanism of the catalytic action of the enzyme for L-3-methylaspartate is not well resolved.In this study,the key amino acid residues affecting the catalysis of L-3-methylaspartate by Asd were identified through a semi-rational design,and the mechanism of the key amino acid residues was analyzed to provide a theoretical basis for the in-depth exploration of the catalytic mechanism of this enzyme and its expanded application.The main findings are as follows:(1)Based on the previous study,molecular modification of laboratory-preserved Asd of Pseudomonas dacunhae,Acinetobacter tandoii and A.radioresistens origin and comparison of their ability to catalyze L-3-methylaspartate,in which Pseudomonas dacunhae Asd(PdAsd)mutant K17A/R37K/V288I catalyzed L-3-methylaspartate twice as well as the previously reported A.radioresistens-derived Asd mutant K18A/R38K/V287I,and therefore P.dacunhae-derived Asd was subsequently selected for the study.(2)Based on molecular docking and substrate channel modification strategies,the key amino acid residues of PdAsd located in the catalytic pocket were identified.The tertiary structure comparison and determination of the specific enzyme activity of the mutants revealed that the arginine residue at position 37 is required for theβ-decarboxylation activity of the enzyme;two point mutations,V288I and T382V,showed significant positive synergistic effects.Based on crystal structure and molecular dynamics simulations to explore the static and dynamic structural changes of the catalytic pocket,the results showed that the pocket cavity in the catalytic center of mutant V288I/T382V was larger and deeper,and the Loop conformation was more suitable for the substrate and bound more stably.(3)The mechanism of action of PdAsd-gated amino acid residues was resolved based on molecular modification.The saturation mutations of gating amino acid residues K17 and R487were carried out to obtain the optimal mutant K17A/R37K/V288I/T382V/R487Q.The results of kinetic parameters showed that two point mutations,K17A and R487Q,could significantly improve the affinity of PdAsd for L-3-methylaspartate.Compared with the mutant V288I/T382V,the substrate affinity(K_m)of K17A/R37K/V288I/T382V/R487Q was increased by 90.7%.The structural analysis results showed that the gating amino acid residues affected the efficiency of the catalytic reaction by controlling the size of the substrate channel entrance and the conformation of the substrate entry. |
