Substrate specificity and enantioselectivity of Est924
It is widely known that bHSLs are promiscuous by nature[15]. For instance, esterase EH1 (PDB: 5JD4) belongs to the bHSL family and demonstrates with a promiscuous substrate specificity [13]. The substrate specificity of Est924 was determined at 30 °C in pH 8.0 buffer by using racemic ethyl (R, S)-2-arylpropionates as substrates. As shown in Figure 1 and Table 1, Est924 could hydrolyze all tested substrates, indicating that it had a wide range of substrates. In the pharmaceutical industry, there is an increasing demand for esterase-mediated chiral resolution of racemic 2-arylpropionate esters due to the high activity and low side effects of the (S)-enantiomers. The enantioselectivity of Est924 was subsequently determined using (R) and (S)-naproxen ethyl ester as substrates. Similar to Est25 [16] and SsoEST1 [9], wild-type Est924 had activity toward (R)-naproxen ethyl ester and (S)-naproxen ethyl ester, with a slight preference for (R) -enantiomer (10%eeR).
Structural analysis of bHSL family esterases
Enzymes with the broad-spectrum substrate applicability generally owe this to their larger active pocket, similar to bHSL family esterases [13]. According to the classification of the Esther database [17], 111 esterase structures of the bHSL family were resolved. We superimposed the crystal structures of Est25 (PDB: 4J7A) and 41 wild-type enzymes of bHSL family (Additional file 1: Figure S2). Three-quarters of bHSL family esterases had highly similar crystal structures, although the lowest sequence similarity observed between 5IQ3 and 3D7R was only 11.7% (Additional file 1: Table S3). The residue L255 of Est25 attracted our attention, because the variant Est25L255W reversed the enantioselectivity of the racemic ketoprofen ethyl ester, resulting in (S)-ketoprofen with 80% eep [18]. Residue L255 was located at the loop between helix 10 and 11 with an isobutyl side chain extension to catalytic serine (Figure 2A). The enantiomer discrimination by enzymes is a very accurate mechanism, which often involves a few amino acids located at the active site. Based on structural information, about three-quarters of the 41 analyzed structures contain the same loop, indicating that it is conserved in the bHSL family (Additional file 1: Table S3). Alternatively, carboxylesterase SsoEST1 has the same residue (L198) as Est25 (L255), but selectively produces (S)-naproxen when racemic naproxen methyl was used as a substrate [9].This indicates that other residues in the active pocket probably control the stereoselectivity of the α carbon. By docking (R)-naproxen ethyl ester into Est25 (Figure 2B), several residues around the naphthalene ring were selected to test whether they played a role in the stereoselectivity of the enzymes, due to the opposite stereoselectivity of Est25 and SsoEST1. As shown in Table 2, the residues at position A2 in all crystal structures were hydrophobic amino acids, and the residues at position B1 exhibited lower conservation (Figure 2C). Since residues at position A2 and position B1 directly extend their side chains into the α carbon of substrates, their low conservation suggested that these residues might be responsible for the different stereoselectivity of enzymes.
Site analysis and rational design of Est924 variants
Est924 can hydrolyze a variety of ethyl 2-arylpropionates, demonstrating the same enantioselectivity as Est25. We simulated the structure of Est924 using the crystal structure of Est25 as a template (Figure 3A). The key residues of Est924 that might control its stereopreferences were analyzed. The selectivity of Est25 mutants with L255W to (S)-ketoprofen ethyl ester was significantly improved, because tryptophan has the largest side chain, which increases the binding steric hindrance of the (R)-enantiomer [11]. Superposition of the Est924 and Est25F72G/L255W crystal structure (5IQ3) revealed that the A203 with a small side chain in Est924 might be responsible for the low enantioselectivity. The Est924 residue was transferred to Est25F72G/L255W by A203W substitution to investigate whether the variant Est924A203W could make (S)-naproxen as the main product (Figure 3B, yellow residues). Similarly, the structure of SsoEst1 was also simulated and superimposed with the Est25 F72G/L255W. The imidazole group of H202 in SsoEst1 occupies the indole ring of W255, which might be the second key residue responsible for the enantioselectivity of the enzymes (Figure 3C, yellow residues). Therefore, G208 at position B1 was also selected as a mutation site to prove our hypothesis. In addition, because the steric hindrance and hydrophobicity of A203 was lower than the phenylalanine in Est25 (F254), SsoEst1 (F197) and Est3 (F203), A203 was also selected for mutation.
The enzyme activity and enantioselectivity of esterases
The enzyme activity and enantioselectivity of esterases were measured using (R, S)-naproxen ethyl ester as substrate by HPLC. Firstly, A203 was mutated to tryptophan according to the structural analysis and results obtained by Kim et al [11]. As predicted, variation of residue A203 significantly impacts enantioselectivity, and the variant M1 (A203W) turned to (S)-selective, producing (S)-naproxen with 60%eep. The variant Est924G208F also reversed the stereoselectivity and hydrolyzed (S)-naproxen ethyl ester with moderate selectivity, yielding (S)-naproxen with 52% eep. These results indicated that both positions (204 and 208) were effective in improving the selectivity of Est924. Variant M2 with A203W and G208F was highly (S)-selective, producing (S)-naproxen with 90% ees. Finally, the variant with mutation at A203 still retained the (S)-enantioselectivity as high as M2, and variant M3 (I202F/A203W/G208F) with 91% ees was obtained.
To clarify why the enantioselectivity of the variants were reversed and increased, we investigated the specific activity (mM/mg/min) for biocatalysis reactions using wild-type Est924, M1, G208F, M2 and M3 with (R)-naproxen ethyl ester and (S)-naproxen ethyl ester as substrates. Est924 showed the lowest enantioselectivity among esterases (Table 3, list 1), which mainly caused similar hydrolysis activity as compared to the enantiomers. M1 retained 90% activity of Est924 on (S)-naproxen ethyl ester, and 15% activity against (R)-enantiomer (Table 3, list 2). Therefore, the enantioselectivity of M1 was reversed and (S)-naproxen was formed as the main product. M3 showed the highest enantioselectivity among the esterases examined. It was almost inactivated in its ability to process (R)-naproxen ethyl ester, but still retained 80% of the activity of Est924 when (S)-naproxen ethyl ester was used as a substrate (Table 3, list 5). Subsequently, the structure of the optimal variant M3 was simulated and docked with (R) and (S)-naproxen ethyl ester, respectively. As shown in Figure 4A, (S)-naproxen ethyl ester fitted well with the active pocket but failed to obtain an effective docking of (R)-enantiomer with M3. Next, we docked (R) and (S)-naproxen ethyl ester into the structure of Est924, as shown in Figure 4B. The naphthalene ring of the (R)-enantiomer pointed to the cavity formed by A203 and G208. The distance between the protonated Nε atom of the active histidine residue and the ester oxygen atom (dNE-O) was usually taken as a geometrical probe to indicate enantioselectivity [20]. The dNE-O of Est924 with (R) and (S)-naproxen ethyl ester were measured in the most favorable binding mode. The ΔdNE-O of (R)-naproxen ethyl ester (3.6Å) was 0.5Å shorter than (S)-naproxen ethyl ester (4.1Å), which corresponded to the slight (R)-stereopreference of Est924. Both M1 (A203W) and M3 had a steric hindrance effect on the (R)-isomer, preventing it from being effectively bound. In addition, the molecular docking results also suggested that (S)-naproxen ethyl ester was not affected by the newly generated steric hindrance from large side chain amino acids in M3. However, mutations reduced the activity pockets of enzymes which were not conducive to the entry of substrates, so their activity on (S)-naproxen ethyl ester were reduced. This suggested that the enantioselectivity reversal of the enzyme was due to steric hindrance effect that made it difficult for the enzyme to bind (R) -enantiomer, which was consistent with the results of specific activity tests.
Resolution of racemic ibuprofen ethyl ester and ketoprofen ethyl ester with M3
In addition to (S)-naproxen, (S)-ibuprofen and (S)-ketoprofen are also widely used. A variety of esterases have been used to produce them through the resolution of corresponding racemic esters [18, 21-25]. Est924 could effectively hydrolyze both racemic ibuprofen ethyl ester and ketoprofen ethyl ester (Table 1). The optimal variant (M3) was tested for enantioselective resolution of these racemic esters. The results showed that M3 preferred to hydrolyze their (S)-conformation esters, generating (S)-ibuprofen (85%ees) and (S)-ketoprofen (87%ees), respectively. To our knowledge, Est924 was the first reported esterase that resolved more than two 2-arylpropionate esters with high enantioselectivity. These results showed that we successfully generated Est924 variants with inverted and highly increased enantioselectivity toward ethyl (S)-2-arylpropionates and provided Est924 mutants with potential application in industrial biocatalysts.