Improving The Stereoselectivity Of An Epoxide Hydrolase PvEH1 By Semi-rational Design

Epoxide hydrolases(EHs,3.3.2.-)can enantioselectively and/or regioselectively catalyze the ring-opening hydrolysis of rac-epoxides,retaining their enantiomers and/or affording their corresponding enantiopure diols.For example,(R)-m-chlorophenyl-ethane-1,2-diol(mCPED)is a building block used in the synthesis of ?3-adrenergic receptor agonist.Previously,a gene encoding PvEHl(pvehl)was cloned from Phaseolus vulgaris,and successfully expressed in E.coli BL21(DE3).In this work,catalytic properties of PvEHl were measured,using m-chlorostyreneoxide(mCSO)as the model substrate.Molecular docking and site-directed mutagenesis were used to analyze the effect of the residue sites lining in substrate binding pocket of PvEH1 on the catalytic properties.Furthermore,after the directed evolution of enantioconvergency was performed,the mechanism of improvement in stereoselectivity was analyzed.Subsequently,the reaction conditions were optimized,followed by the gram-scale preparation of(R)-mCPED.After being homologically modeled and optimized,the three-dimensional structure of PvEHl was docked with(R)-or(S)-mCSO to confirm interaction with 23 residue sites located in the substrate binding pocket of PvEHl.According to the results of multiple sequence alignment with five EHs which were reported to have high enantioconvergency towards rac-mCSO,substitute amino acids of 16 sites were predetermined.The remained seven sites were all replaced by leucine,respectively.Among 23 single-site mutants constructed in this work,five ones,pvEHlV106I,PvEH1M129L,PvEH1M160A,PvEH1M175I and PvEH1S178T,displayed significantly increased enzyme activity,while four ones,PvEH1w102L,PvEH1L105I,PvEH1Y149L and PvEH1P184L,with markedly improved enantioconvergency.PvEH1M129L has the highest enzyme activity which was 1.6 folds higher than that of PvEH1,while PvEH1W102L owned the highest enantioconvergency which was 79.6 times that of PvEHl.The above-metioned residue sites with a positive effect on the catalytic propieties of PvEHl were divided into two types:five sites(106,129,160,175 and 178)increasing catalytic activity and four sites(102,105,149 and 184)increasing enantioconvergency.Nine site-directed saturation mutagenesis libraries were constructed and screened to figure out the best substitute amino acid of each site.The results showed that,the most preferred substituted amino acids at each residue sites which increasing catalytic activity were 1106,L129,A160,1175 and T178,respectively.Five-site random combinatorial mutagenesis library to improve the catalytic activity was constructed and screened,where mutant PvEH1Z4(including V106I,M160A,M175I and S178T)was obtained.The catalytic activity of PvEH1Z4 was 2.1-fold higher than that of PvEH1.The most preferred substituted amino acids at each residue sites which increasing enantioconvergency were L102,1105,L149 and W184,respectively.Then,using PvEH1Z4 as a template,four-site random combinatorial mutagenesis library to improve the enantioconvergency was constructed and screened,where mutant PvEH1Z4X4-59(including L105I,V106I,M160A,M175I,S178T and P184W)was obtained.Its catalytic activity and enantioconvergency were 2.6-fold and 92.6-fold higher than those of PvEH1.The regioselectivity coefficients and enantiomeric ratio of PvEHl and its mutants were determined.Accidentally,W102L could inproved the enantioselectivities of mutants,for example,PvEH1W102L displayed a high enantioselectivities towards mCSO and p-chlorostyreneoxide(pCSO),with enantiomeric ratios of 30 and 89.PvEH1W102L was applied into kinetic resolution of rac-pCSO.The molecular mechanism was analyzed by molecular docking and molecular dynamics simulation.The result showed that,the replacement of Trp102 by Leu made enzyme bind with(R)-p-chlorostyreneoxide(pCSO)harder than before,hence improved enantioselectivity of PvEH1W102L towards pCSO.In addition,regioselectivity coefficients of PvEH1Z4X4-59 were all over 96%.After the residue site 184 was substituted by W and residue site 105 was substituted by L,the position of nucleophilic attack from PvEH1Z4X4-59 is switched from C? to C? of the oxirane ring of(S)-mCSO.On the other hand,nucleophilic attack of the oxirane ring of(R)-mCSO remained largely unaltered in the PvEH1Z4X-59,which creates an enantioconvergency towards rac-mCSO.The initial reaction concentration and time of kinetic resolution of rac-pCSO catalyzed by E.coli/pvehlW102L in buffer system were optimized.After four-hour reaction,the yield of(R)-pCSO in the kinetic resolution was 45.62%(96.3%ees),when the yield of(R)-p-chlorophenyl-1,2-ethanediol was 50.91%(90.26%eep).In addition,the concentration of rac-mCSO and reaction medium and was optimized.Hydrolysis of 2.7 g rac-mCSO was carried out on the basis of adding 5%Tween 20,with a space time yield of 6.32g·h-1·L-1.The 2.51g(R)-mCPED has an eep of 93.1%,a yield of 83.7%.