Gene Screening,Molecular Modification And Function Research Of L-TA With High Stereoselectivity

Droxidopa（L-threo-DOPS）is a new anti-Parkinson’s disease drug.It was approved to improve gait stiffness and orthostatic dizziness caused by Parkinson’s disease.Currently,L-threo-DOPS is mainly produced by chemical methods.The chemical synthesis method mainly uses 3,4-dihydroxybenzaldehyde as a starting material,and obtains chiral droxidopa through an eight-step reaction such as addition reaction,esterification reaction and chemical resolution with a 11%total yield.The steps of this method are complicated,involving chemical chiral resolution,adding/deprotecting groups,using a large number of organic reagents,strong acids and alkalis,and other harsh reaction conditions.The environment is unfriendly and there are a large number of optical isomers as by-products.With the development of the integration of biotechnology and engineering technology,it will become possible to apply biocatalytic technology to large-scale preparation of L-threo-DOPS.Early investigation found that L-threonine aldolase（L-TA）was a key enzyme that drive the efficient biosynthesis of L-threo-DOPS from 3,4-dihydroxybenzaldehyde and acetaldehyde by reversible condensation in vitro.However,at present,there has been a problem of poor stereoselectivity ofβ-hydroxyl to synthesize L-threo-DOPS with L-TAs.The C_β-diastereoselectivity（de value）of L-TAs is a key factor in determining whether it can adapt to industrial production.In order to obtain L-TAs with high de values,a new L-TA（named Sz-1-2）was obtained from gut microbiota of black bears using NCBI database alignment analysis technology and fusion protein expression technology.The optimized conditions of Sz-1-2 were systematically screened.Further based on bioinformatics analysis,the active pocket residues were molecularly modified to obtain variant R318C with increased de value.Saturated mutation was performed on the increased 318 position.The reasons for the improvement of de values of the mutants R318M,R318V and R318L were investigated by using the three-dimensional structure homology modeling method and molecular docking method.This study expanded the natural enzyme library of L-TAs and provided new resources for large-scale preparation of L-threo-DOPS using biocatalytic technology.The main research contents and results are as follows:（1）Based on the laboratory early bear gut microgenomics database,we obtained a new a new L-TAs（named Sz-1-2）.（1）Homology and phylogenetic tree analysis indicated that Sz-1-2 belongs to aldolase family,which has PLP dependent.（2）SDS-PAGE and MALDI-TOF-MS mass spectrometry analysis showed that the molecular weight of Sz-1-2 was consistent with the theoretical prediction of 39.68 k Da.（3）The catalytic activity of Sz-1-2 showed that the specific activity of Sz-1-2 for L-threonine was higher than that of most reported L-TAs,which met the preliminary conditions for the synthesis of L-threo-DOPS.From the high synthetic activity of Sz-1-2,Sz-1-2 is a promising enzyme for the synthesis of droxidopa.（4）The de value of the synthesized L-threo-DOPS was found to be 21%for the wild type Sz-1-2.Compared with the currently reported wild-type L-TA（de value=14%）,it has a higher de value,which has a significant advantage in biosynthetic L-threo-DOPS.（2）The optimal conditions for the synthesis of L-threo-DOPS from Sz-1-2 were systematically studied.（1）The enzymatic kinetic study of Sz-1-2 found that Sz-1-2 has a smaller K_mvalue and a larger(k_cat/K_m).At the same time,Sz-1-2 is a rare example with a higher affinity for L-threonine,indicating that it has a higher probability of high diastereoselective synthesis of L-threo-DOPS.（2）The optimal reaction conditions obtained after the optimization of the conditions are:the substrate is 1 M glycine,60m M 3,4-dihydroxybenzaldehyde,the coenzyme is 50μM pyridoxal phosphate,and the reaction is performed in p H 7.5 PBS buffer for 4 h.L-threo-DOPS was synthesized using the optimal reaction conditions.The diastereoselectivity of Sz-1-2 after optimization was 30.2%,which was 43.8%higher than the 21%before optimization.The study of enzymatic properties provides data references for the subsequent research and application of Sz-1-2.（3）Based on the bioinformatics analysis,this article carried out molecular modification of the active pocket residues of Sz-1-2,obtained ten Sz-1-2 mutants,and among the ten mutants,Arg318 with significantly increased de value was selected for saturation mutation.（1）The research on the activity and de value of ten mutants in the first round of mutation showed that only mutant R318C showed an increase de value for the synthesis of L-threo-DOPS,which was about 1.7 times that of wild type,reaching53.6.%,so Arg318 was selected as the second round of saturated mutation site.（2）The results of the de value study of the second round of Arg318 saturated mutants found that most of the Arg318 mutants showed improved diastereoselectivity.Mutants R318M,R318V,and R318L showed significantly enhanced de values（77.1%,79.9%,and 84.9%,respectively）in the synthesis of L-threo-DOPS.Among which the R318L mutant had the best diastereoselectivity,it is 2.8 times that of wild-type Sz-1-2 and 1.5 times that of the best enzyme（55.4%de）currently reported.Obtaining mutants with increased de value laid the foundation for the large-scale preparation of L-threo-DOPS by biocatalysis technology.（4）We used SWISS-MODEL to model the homologous three-dimensional structure of wild-type Sz-1-2 and mutants R318M,R318V and R318L,and the substrate and coenzyme complex were docked to the active center using CDOKER.The results show that for the wild-type Sz-1-2,the residues Arg318 and Arg172 interacted strongly with the carboxyl group of L-threo-DOPS/PLP complex,which made the complex adopt a conformation with its phenolic group pointing to His128.However,when the residue Arg318 was substituted by those nonpolar amino acids,the interaction between Arg318 and the carboxyl group of L-threo-DOPS/PLP complex disappeared and Arg172could still function,leading to a distinct binding mode of the complex in which the phenolic group of the complex was far away from His128 and directed to those mutated sites 318 with nonpolar amino acids.The obviously different conformations of L-threo-DOPS/PLP complex in between the wild-type Sz-1-2 and its mutants R318M,R318V,R318L might contribute to the significant improvement of diastereoselectivity.These results suggested that Arg318 was very important to the diastereoselectivity of Sz-1-2.