Construction Of A Biosynthesis Route Of L-2-aminobutyric Acid And Molecular Modification Of The Key Enzyme

L-2-Aminobutyric acid(L-ABA)is an important unnatural amino acid that is a key chiral precursor for the production of the anti-epileptic levetiracetam and brivaracetam and the antituberculosis ethambutol.An biosynthesis of L-ABA combines deamination of L-threonine and amination of 2-oxobutyric acid to form L-ABA.There are some technical problems,such as the requirement for amino donors,the need for an external cofactor(NADH)regeneration system,uncontrollability of reaction equilibrium and formation of the byproduct L-alanine.These involute processes of L-ABA synthesis have resulted in prohibitive prices of the drugs using L-ABA as a key chiral precursor,and a cost-effective synthesis route that avoids the above mentioned obstacle is anticipated.To solve the the problems of high energy consumption,high cost and low efficiency in production of L-ABA using L-threonine as substrate.A new green biosynthetic route was designed consisting of two steps: L-glutamate is converted into(2S,3S)-3-methylaspartate by glutamate mutase(EC 5.4.99.1,GM),followed by irreversible ?-decarboxylation of 3-methylaspartate by L-aspartate-?-decarboxylase(E.C.4.1.1.12,Asd)to form L-ABA.This route had the advantages of simple production process,high purity of product,low cost of raw material L-glutamate,which greatly reduced the production cost of related drugs using LABA as a key chiral precursor,and had a great application prospect.The main results were listed as follows:(1)The molecuar modification of gate of substrate tunnel and active site of Asd was performed based on sequence alignment and enzyme structure analysis to improve the ?-decarboxylation activity towards 3-methylaspartate.L-aspartate is a natural substrate for Asd,However,the activity of wide type(WT)Ar Asd towards 3-methylaspartate was not detected at all.Based on sequence alignment and structural analysis,the gate of substrate tunnel of Asd from Acinetobacter radioresistens(Ar Asd)was identified and rationally broadened and the conformation of the substrate in the active site was changed.Compared to WT Ar Asd,K18A/V287 I showed a specific activity of 18.5±0.12 U/mg towards 3-methylaspartate.The product L-ABA catalyzed by the K18A/V287 I from 3-methylaspartate was confirmed by HPLC and LC-MS/MS determination.The yield of L-ABA catalyzed by the K18A/V287 I from 3-methylaspartate reached 99%.Molecular docking and molecular dynamics simulations were carried out to analyze the reasons for the altered substrate specificity of K18A/V287 I.In K18A/V287 I,the gate of the substrate tunnel was broadened and the orientation of methyl and carboxyl groups of 3-methylaspartate at C? was inverted compared with that of WT Ar Asd,which was beneficial to help Asd catalyze ?-decarboxylation of 3-methylaspartate.(2)The molecular modification of the residue controlling the energy levels of the starting states of Ar Asd was performed to alter the substrate perference of Ar Asd and improve the catalytic efficiency.The sequence alignment and structure analysis of Ar Asd indicated that the Arg38 of Ar Asd was identified as a key residue controlling the strain of the internal aldimine and regulating the energy levels of the starting states of Ar Asd during catalysis.The mutant K18A/R38K/V287 I was obtained using K18A/287 I as a template by site-mutagenesis.Compared to the K18A/V287 I,the K18A/R38K/V287 I increased the kcat for 3-methylaspartate(3.0-fold)more than that of L-Asp(1.5-fold)and was more preference to accommodate 3-methylaspartate.The molecular dynamics simulations showed that the K18A/R38K/V287 I reduced the number of hydrogen bonds between the Lys38 and PLP compared with the K18A/V287 I,which may change the conformation of the internal aldimine and raise the energy level of the internal aldimine leading to enhance the catalytic efficiency.Compare to K18A/V287 I,K18A/R38K/V287 I may break the hydrogen bond network between substrate and Arg496,Arg374,and Asn 256 leading to a decreasing the affinity toward all substrates,especially for L-Asp.(3)This was the first time for L-ABA synthesis through straightforward ?-decarboxylation of L-glutamate by using GM and Asd.In this study,by comparing enzymatic properties of the reported GM,a subunit-fused GM with simplified subunit structure and kinetic properties was selected to convert L-glutamate into 3-methylaspartate.The optimal reaction conditions for the production of L-ABA were performed and as follows: the temperature was 37?,the p H was 6.6,the ratio of GM and K18A/V287 I was 1:1,the concentration of GM and K18A/V287 I was respectively 4 mg/ ml,the pyruvate was 0.05 m M and the DTT was 0.1m M.The yield of L-ABA was 90% after the reaction for 4 h.(4)The production of L-ABA was improved by introducing the GM reactivating factor Mut L into the reaction system of GM coupled with Asd,which recovered the activcity of the inactivated GM.GM was easily inactivated by incubating with substrate or Adenosylcobalamin for a long time,Mut L can help the inactived GM to recover its activity.In the late stage of the reaction in GM coupled with Asd,Mut L recovered the activity of the inactivated GM,which lead to improve the yield of L-ABA.The yield of L-ABA was 97% after introducing Mut L into the reaction system.