Improving Enantioselectivity Of Epoxide Hydrolase AuEH2 For Ortho-substituted Aromatic Epoxides

Enantiopure epoxides and vicinal diols are important multifunctional chiral building blocks,which are widely used in medicines,fine chemicals and other biologically active substances.Epoxide hydrolases（EHs）can specifically catalyze racemic epoxides,retaining chiral ones and generating chiral vicinal diols.Compared with chemical catalysis,EH has the advantages of broad substrate spectrum,independent on cofactors and mild reaction conditions,and is a potential biocatalyst.An epoxide hydrolase AuEh2 from Aspergillus usamii was discovered in the early stage of our laboratory,and its catalytic properties were studied and the crystal structure was obtained.According to reports,most EHs have unsatisfactory or inactive catalytic properties for ortho-substituted aromatic oxide.To improve AuEh2’s enantioselectivity for the kinetic resolution of ortho-substituted styrene oxide,directional modification of AuEh2 was carried out.（1）The AuEh2 crystal structure（PDB:6IX4）obtained earlier in our laboratory contains two AuEh2 subunits and belongs to theα/β-hydrolase folding superfamily.The single subunit of AuEh2 contains three typical m EH domains:N-terminal anchor domain,cap domain andα/βdomain.The active center contains the catalytic triad（D191-H369-E343）in theα/βdomain and Y249 and Y312 in the cap domain.Based on the analysis of AuEh2 crystal structure,10non-conservative amino acid residues in the substrate binding pocket were selected as the pseudo-mutation sites,and the mutant library was constructed using the site-directed saturation mutagenesis method.（2）Using racemic ortho-fluoro styrene oxide（rac-o-FSO）as a substrate to screen the mutant library,the results showed the E values of A214V（E=64）,A250I（E=108）,N315M（E=32）,R322T（E=32）and L344V（E=38）were increased compared with the enantiomeric ratio（E=31）of wild-type AuEh2.Using p ET-28a-Aueh2^A250I as the template,double-site combinatorial mutations were performed with the mutants above,respectively.The E value of the optimal mutant A250I/L344V was increased to 180 and its activity was 82 U·g^-1 wet cell.The kinetic resolution of 400 mmol·L^-1 rac-o-FSO was performed by 100 mg·m L^-1 E.coli/Aueh2^A250I/L344V,obtaining（S）-o-FSO with 97.7%enantiomeric excess（ee）and 46.7%yield within 6 h.（3）Using ortho-trifluoromethyl styrene oxide（rac-o-TFMSO）as a substrate to screen the mutant library,the results showed that several mutants at sites 195,216,315,322,344,and 345with significantly improved enantioselectivity were screened.The best mutant was K195W,whose E value was increased from 8 to 37.Using p ET-28a-Aueh2 as the template,the above-mentioned sites were subjected to combinatorial active-site saturation test.The best mutant R322V/L344C was obtained,whose E value was increased to 83 and its activity was 67 U·g^-1wet cell.The kinetic resolution of 200 mmol·L^-1 rac-o-TFMSO was performed with 80 mg·m L^-1 E.coli/Aueh2^R322V/L344C under ice bath,obtaining（S）-o-TFMSO with 98.4%ee_s and 49.3%yield within 5.5 h,and the E value was increased from 83 to>200.（4）Using ortho-methylstyrene oxide（rac-o-MSO）as the substrate to screen the mutant library,the results showed that compared with AuEh2（E=4）,only A250Q（E=8）and A250I（E=11）,A214V（E=6）,S247Q（E=7）,S247F（E=9）,S247W（E=9）had a slight increase.Using p ET-28a-Aueh2^A214Vas the template,double-site combinatorial mutations were carried out with S247Q,S247F and S247W respectively.Among the obtained mutants,the E value of A214V/S247Q was increased to 28,and the activity was 55 U·g^-1 wet cell.（5）To gain insight into the source of enhanced selectivity of AuEh2^A250I/L344V and AuEh2^R322V/L344C to specific substrates,the crystal structure of AuEh2^A250I/L344V was obtained and analyzed.At the same time,using AuEh2 crystal structure as the template,homology modeling of the three-dimensional structure of AuEh2^R322V/L344C was performed.And those structure were docked with their respective substrates.The results showed that the distance between D191 of AuEh2^A250I/L344V and C_βof（R）-o-FSO was closer compared with AuEh2.According to the proximity attack theory,the C_βwas more susceptible to nucleophilic attack by D191,causing AuEh2^A250I/L344V to prefer hydrolyzing（R）-o-FSO.Similarly,the distance between D191 of AuEh2^R322V/L344C and C_βof（R）-o-TFMSO was closer than AuEh2,so AuEh2^R322V/L344C hydrolyzes（R）-o-TFMSO more preferentially and retaining more（S）-o-TFMSO.