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Directed modification of a novel epoxide hydrolase from Phaseolus vulgaris to improve its enantioconvergence towards styrene epoxides
Catalysis Communications 87 (2016) 32–35
Contents lists available at ScienceDirect
Catalysis Communications journal homepage: www.elsevier.com/locate/catcom
Short communication
Directed modification of a novel epoxide hydrolase from Phaseolus vulgaris to improve its enantioconvergence towards styrene epoxides Hui-Hua Ye a,1, Die Hu b,1, Xiao-Ling Shi a, Min-Chen Wu c,⁎, Chao Deng c, Jian-Fang Li d,⁎ a
School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, PR China Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China Wuxi Medical School, Jiangnan University, Wuxi 214122, PR China d School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China b c
a r t i c l e
i n f o
Article history: Received 1 July 2016 Received in revised form 17 August 2016 Accepted 25 August 2016 Available online 26 August 2016 Keywords: Directed modification Epoxide hydrolase Phaseolus vulgaris Enantioconvergence Styrene epoxides
a b s t r a c t To improve the enantioconvergence of an epoxide hydrolase from Phaseolus vulgaris (PvEH1) towards styrene epoxides, its directed modification was performed based on the rational design by using the molecule docking simulation and the multiple alignment. The single- and multi-site mutation genes of pveh1 were constructed as designed theoretically by PCR, and expressed in E. coli BL21(DE3), respectively. Among all PvEH1 mutants tested, a three-site mutant, PvEH1L105I/M160A/M175I, was selected having the highest activity of 10.66 U/g wet cell and the best regioselectivity (αS N 99%, βR = 86.4%), by which rac-1a was transformed into (R)-1b with 87.8% enantiomeric excess (eep), much higher than that (33.6% eep) by PvEH1. Furthermore, it completely hydrolyzed rac2a–5a into (R)-2b–5b with 52.3–70.9% eep. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Epoxide hydrolases (EHs) can catalyze the kinetic resolution or enantioconvergent hydrolysis of epoxides, retaining epoxide enantiomers or convergently transforming into diols. The products are versatile chiral synthons for the synthesis of highly value-added pharmaceuticals, agrochemicals and fine chemicals [1,2]. The resolution has an intrinsic limitation: the maximum yield of any enantiomer never can exceed 50% [3]. Comparatively, the enantioconvergent hydrolysis, by enzymatic or chemoenzymatic catalysis, can afford diols with high eep and theoretical yield of 100% [4]. The enantioconvergent hydrolysis of epoxides by single EH is an ideal process, but few naturally occurring EHs possess the opposite regioselectivity towards (S)- and (R)-enantiomers [5]. As to plant EHs, only ones from Solanum tuberosum [6] and Vigna radiata [7,8] exhibited good enantioconvergence. Owing to the shortage of enantioconvergent EHs and their advantages in preparing chiral diols, it is desirable to explore more EHs. On the other hand, the enantioconvergent modification of EHs has been performed over the last decade [9]. For example, after five rounds of iterative saturation mutagenesis on Aspergillus niger M200 EH, the best mutant was selected, by which racemic styrene oxide (rac-SO) and para-chlorostyrene oxide (pCSO) were transformed into (R)-diols with over 70% eep [10]. The ⁎ Corresponding authors. E-mail addresses: [email protected] (M.-C. Wu), [email protected] (J.-F. Li). 1 Hui-Hua Ye and Die Hu, the two first authors, contributed equally to this work.
http://dx.doi.org/10.1016/j.catcom.2016.08.036 1566-7367/© 2016 Elsevier B.V. All rights reserved.
exploration and modification of EHs promoted the understanding on the mechanism of enantioconvergent hydrolysis [11]. Recently, a PvEH1-encoding gene (pveh1, GenBank: KR604729) was cloned from P. vulgaris and expressed in E. coli BL21(DE3) in our lab. The enantioconvergent hydrolysis of styrene epoxides by PvEH1 was carried out (Fig. 1). However, the regioselectivity coefficients (αS = 91.1%, βR = 53.3%) towards (S)- and (R)-1a was insufficient, resulting in a low purity of (R)-1b (only 33.6% eep). In this work, to enhance the βR and activity of PvEH1, nine mutant genes were constructed as designed theoretically by PCR and expressed in E. coli BL21, respectively. Using the whole cells of E. coli transformants as catalysts, the properties of PvEH1 and its mutants were characterized and compared.
2. Experimental 2.1. Materials The recombinant plasmid, pET-28a-pveh1, was constructed and preserved in our lab. PrimeSTAR HS DNA polymerase and endonuclease Dpn I (TaKaRa, Dalian, China) were applied for the construction of pveh1 mutants. E. coli BL21(DE3) (Novagen, Madison, WI) was used for the expression of EHs. Rac-SO (1a), (S)- and (R)-1a (Energy, Shanghai, China) were used for the enantioconvergence screening, while rac1a derivatives (2a–5a) (Fig. 1), synthesized by our lab, for the substrate spectrum assay.
H.-H. Ye et al. / Catalysis Communications 87 (2016) 32–35
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Fig. 1. The enantioconvergent hydrolysis of styrene epoxides into (R)-diols by PvEH1.
2.2. MD simulation and multiple alignment
2.5. Catalytic properties of EHs
Based on the crystal structure of an S. tuberosum EH (PDB: 2CJP), the three-dimensional (3-D) structure of PvEH1 was modeled by MODELLER 9.11 program (http://salilab.org/modeller/). Meanwhile, the 3-D structure of (R)-1a was handled using a ChemDraw Ultra 12.0 software (www.cambridgesoft.com/). The interaction of PvEH1 with (R)-1a, towards whom the βR of PvEH1 is lower, was predicted by MD simulation by AutoDock 4.2 program (http://autodock.scripps.edu) to locate the most suitable binding position and orientation. According to the conformation of PvEH1 docking with (R)-1a, residues in proximity to (R)-1a within 8 Å were confirmed by PyMol software (http://pymol.org/). Plant EHs were searched at the NCBI website (http://blast.ncbi.nlm. nih.gov/) by BLAST server. Then, the multiple alignment of PvEH1 with three EHs having excellent enantioconvergence was performed by ClustalW2 program (http://www.ebi.ac.uk/Tools/msa/). According to the properties of non-conserved residues in proximity to (R)-1a within 8 Å and their positions on PvEH1 3-D structure, several residues were selected to be replaced with the corresponding and frequently emerging ones of S. tuberosum and V. radiata EHs, forming a series of PvEH1 mutants.
The enantioconvergence of EH towards rac-1a was assayed by incubating 475 μL cell suspension and 25 μL rac-1a (10 mM) at 25 °C. After complete hydrolysis, the product was extracted with 1 mL ethyl acetate having 1 mM n-hexanol (internal standard) and analyzed by GC (Table S2). Using (R)- or (S)-1a instead of rac-1a, the regioselectivity coefficients of mutants with enhanced eep were measured. In addition, the substrate spectrum of the best mutant was assayed by replacing rac1a with rac-2a–5a, respectively, by HPLC (Table S2).
2.3. Construction of E. coli transformants The site-directed mutagenesis of pveh1 was conducted by whole-plasmid PCR technique. PCR primers were designed (Table S1). The single-site mutation genes were amplified from pET-28a-pveh1 as following conditions: 30 cycles at 98 °C for 10 s, 55 °C for 15 s and 72 °C for 8 min. The PCR products were digested by DpnI and transformed into E. coli BL21, respectively, forming five E. coli transformants. Similarly, double-site mutation genes, pveh1L105I/M160A and pveh1L105I/M175I, from pET-28a-pveh1L105I were used to construct E. coli transformants, E. coli/pveh1L105I/M160A and E. coli/pveh1L105I/M175I, while pveh1M160A/M175I and pveh1L105I/M160A/M175I separately from pET-28a-pveh1M160A and pET-28a-pveh1L105I/M160A were applied to construct E. coli/pveh1M160A/M175I and E. coli/pveh1L105I/M160A/M175I.
3. Results and discussion 3.1. Selection of mutation sites The highest primary structure identities, analyzed by BLAST server, of PvEH1 with EHs from V. radiata [7], Medicago truncatula [12] and Glycine max (XP_003554633) are 85.7, 81.1 and 81.0%, indicating that PvEH1 was a novel EH. It belongs to the α/β-hydrolase superfamily having two characteristic regions, α/β-hydrolase and lid domains. Its catalytic triad is D101-H299-D264 and two proton donors in the lid are Y150 and Y234 (Fig. S1). According to the docked conformation of PvEH1 with (R)-1a (Fig. 2a), 50 amino acids of PvEH1 in proximity to (R)-1a within 8 Å were located by PyMol software. Based on the multiple alignment of PvEH1 with three plant EHs (VrEH1, VrEH2 and StEH) having excellent enantioconvergence (Fig. 2c), 28 absolutely conserved residues and 14 relatively conserved ones (the residues of PvEH1 were the same as those of two in other three EHs) were excluded. Thus, the remaining eight non-conserved residues were used as the mutant candidates, from which five non-conserved ones, L105, M129, M160, M175 and H267, were selected based on their positions on PvEH1 3-D structure (Fig. 2b) and their distances to catalytic residues and substrate (R)-1a (Table S3). As shown in Fig. 2b, the five selected residues are located near the active center and around (R)-1a. 3.2. Enantioconvergence of EHs towards rac-1a
2.4. Enzyme activity assay After E. coli transformant was induced by 0.5 mM IPTG at 20 °C for 10 h, cells were suspended in 100 mM Na2HPO4-NaH2PO4 buffer (pH 7.0). Cell suspension (1 g wet cell per 10 mL) was used as a catalyst. EH activity was analyzed by HPLC. In brief, 450 μL cell suspension was mixed with 50 μL rac-1a (final concentration of 10 mM). After incubation at 25 °C for 10 min, the reaction was stopped by adding 1 mL methanol. The sample was analyzed using a Waters e2695 system (Waters, Milford, MA) equipped with a C18 column. The mobile phase of methanol/H2O (7:3, v/v) was used at a flow rate of 0.8 mL/min, and monitored at 220 nm using a Waters 2489 UV–Vis detector. One unit (U) of EH activity was defined as the amount of enzyme hydrolyzing 1 μmol rac-1a per minute.
The eep, enantiomeric ratio (E) value and activity of EH towards rac-1a were measured (Table 1). The single-site mutants were constructed by separately replacing residues (L105, M129, M160, M175 and H267) of PvEH1 with the frequently emerging ones (I, L, A, I and Y) of VrEH1, VrEH2 and StEH. The eep values by PvEH1L105I, PvEH1M160A and PvEH1M175I increased by 128, 46 and 58%, respectively, over that (33.6% eep) by PvEH1. In addition, their activities were 3.03-, 1.46- and 4.26-fold higher than that (1.61 U/g wet cell) of PvEH1. As shown in Fig. S2, the expression levels of PvEH1 and its mutants displayed no difference, indicating that the activity improvement of three mutants was not expression amount increase. Furthermore, PvEH1 and PvEH1L105I were isolated from E. coli cells and purified. The specific activity of purified PvEH1L105I was 6.49 U/mg protein, which was 2.82-fold higher than that (2.30 U/mg protein) of purified PvEH1 close to 3.03 folds calculated based on E. coli whole cells. Then,
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H.-H. Ye et al. / Catalysis Communications 87 (2016) 32–35
Fig. 2. (a) The interaction of PvEH1 with (R)-1a predicted by MD simulation. The catalytic triad of PvEH1 is D101-H299-D264 and two proton donors are Y150 and Y234. (b) Five selected residues, L105, M129, M160, M175 and H267, are located near the active center and around (R)-1a. (c) Multiple alignment of PvEH1 with VrEH1, VrEH2 and StEH. Fifty residues of PvEH1 in proximity to (R)-1a within 8 Å are marked with stars.
according to the different assembly of three mutation sites, four multi-site mutants were constructed. As expected, eep values by PvEH1L105I/M160A, PvEH1L105I/M175I and PvEH1L105I/M160A/M175I were 81.0, 84.1 and 87.8%, while their activities were 4.83, 8.87 and 10.66 U/g wet cell. All eep values Table 1 Catalytic properties of PvEH1 and its mutants towards rac-1a. Epoxide hydrolase
eepa (%)
E valueb
Relative activity (%)
PvEH1 L105I M129 L M160 A M175I H267Y L105I/M160 A L105I/M175I M160 A/M175I L105I/M160 A/M175I
33.6 ± 1.3 76.5 ± 3.2 39.9 ± 1.5 49.2 ± 2.0 53.2 ± 2.5 – 81.0 ± 3.6 84.1 ± 4.1 56.9 ± 2.7 87.8 ± 4.4
1.5 ± 0.1 3.2 ± 0.3 2.1 ± 0.2 2.0 ± 0.1 3.0 ± 0.1 – 2.2 ± 0.1 2.0 ± 0.2 1.8 ± 0.1 3.6 ± 0.2
100 303 ± 14 94 ± 6 146 ± 9 426 ± 17 50 ± 4 300 ± 10 551 ± 26 257 ± 11 662 ± 33
of (R)-1b and (S)-1b. a eep = [(Rp − Sp)/(Rp + Sp)] × 100%. Rp and Sp are the final concentrations. b E = ln (R/R0)/ln (S/S0). R and S are the final concentrations of (R)-1a and (S)-1a, while R0 and S0 the initial concentrations.
by three mutants were higher than those (70.1 and 77.0% eep) by AnEHH:12-A1 and Kau2 [10,13]. 3.3. Regioselectivity coefficients of EHs The regioselectivity coefficients of PvEH1 and its four mutants towards (S)- and (R)-1a were assayed (Fig.3). The βR values of PvEH1L105I, PvEH1L105I/M160A, PvEH1L105I/M175I and PvEH1L105I/M160A/M175I were enhanced from 53.3% to 77.8, 83.0, 84.4 and 86.4%, respectively, while the αS values of four mutants exceeded 99%. The regioselectivity of PvEH1L105I/M160A/M175I was higher than that (αS = 83%, βR = 68%) of mbEH A (renamed VrEH1) [14], but still lower than that (αS = 98%, βR = 93%) of StEH [6]. Usually, the increase of one coefficient would accompany with decrease of the other one, such as the directed evolution of AnEH [10] and Kau2 [13,15]. To our knowledge, it was rare that both αS and βR were synchronously enhanced as reported here. 3.4. Substrate spectrum of PvEH1L105I/M160A/M175I The substrate spectrum of PvEH1L105I/M160A/M175I was assayed (Table 2). The eep values of (R)-2b and 4b catalyzed by the best mutant were
H.-H. Ye et al. / Catalysis Communications 87 (2016) 32–35
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Analysis of the single-site mutants verified that three residue sites, L105, M160 and M175, have notable effects on the eep and activity of PvEH1 towards rac-1a. Then, the four multi-site mutants were constructed according to the different combination of mutation sites. Among all PvEH1 mutants tested, PvEH1L105I/M160A/M175I has the best regioselectivity and highest activity, by which rac-1a was transformed into (R)-1b with high enantiomeric purity. In addition, it showed a broader substrate spectrum. Our present work laid a solid foundation for the modification of this type of EHs to improve their enantioconvergence and activities. Acknowledgment
Fig. 3. The regioselectivity coefficients of a wild-type PvEH1 and its four mutants, PvEH1L105I, PvEH1L105I/M160A, PvEH1L105I/M175I and PvEH1L105I/M160A/M175I, towards (S)and (R)-1a.
increased from 50.3 and 51.4% to 64.7 and 70.9%, respectively. Additionally, PvEH1 L105I/M160A/M175I catalyzed the hydrolysis of rac-3a and 5a into (R)-3b and 5b, respectively, with 52.3 and 69.7% ee p , much higher than those (14.7 and 1.0% eep) by PvEH1. The activity of PvEH1L105I/M160A/M175I was approximately equal to that of PvEH1 towards the same substrate (2a–5a). However, both of them had higher activities towards chloro-substituted styrene epoxides than those towards nitro-substituted ones.
4. Conclusions Based on the rational design, the modification of PvEH1 was performed, which have improved its properties towards styrene epoxides.
Table 2 The substrate spectra and activities of PvEH1 and PvEH1L105I/M160A/M175I. Substrate
1a 2a 3a 4a 5a
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.catcom.2016.08.036. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
PvEH1L105I/M160A/M175I
PvEH1
This work was financially supported by the National Nature Science Foundation of China (No. 21676117), the Fundamental Research Funds for the Central Universities of China (No. JUSRP51412B), and the Postgraduate Innovation Training Project of Jiangsu, China (Nos. KYLX15_1151 and _1190). We are grateful to Prof. Xianzhang Wu (School of Biotechnology, Jiangnan University) for providing technical assistance.
eep (%)
Activity (U/g)
eep (%)
Activity (U/g)
33.6 ± 1.3 50.3 ± 3.3 14.7 ± 0.7 51.4 ± 2.4 1.0 ± 0.1
1.61 ± 0.08 1.30 ± 0.06 4.26 ± 0.16 7.28 ± 0.27 5.83 ± 0.26
87.8 ± 4.4 64.7 ± 1.4 52.3 ± 2.6 70.9 ± 3.5 69.7 ± 3.3
10.66 ± 0.48 1.74 ± 0.08 4.37 ± 0.17 8.50 ± 0.43 5.16 ± 0.23
[12]
[13] [14] [15]
E.Y. Lee, Biotechnol. Lett. 30 (2008) 1509–1514. M. Wang, T. Si, H.M. Zhao, Bioresour. Technol. 115 (2012) 117–125. W.J. Choi, Appl. Microbiol. Biotechnol. 84 (2009) 239–247. M. Kotik, A. Archelas, R. Wohlgemuth, Curr. Org. Chem. 16 (2012) 451–482. N. Bala, S.S. Chimni, Tetrahedron Asymmetry 21 (2010) 2879–2898. M.I. Monterde, M. Lombard, A. Archelas, A. Cronin, M. Arand, R. Furstoss, Tetrahedron Asymmetry 15 (2004) 2801–2805. Q.Q. Zhu, W.H. He, X.D. Kong, L.Q. Fan, J. Zhao, S.X. Li, J.H. Xu, Appl. Microbiol. Biotechnol. 98 (2014) 207–218. Y.W. Wu, X.D. Kong, Q.Q. Zhu, L.Q. Fan, J.H. Xu, Catal. Commun. 58 (2015) 16–20. E.Y. Lee, M.L. Shuler, Biotechnol. Bioeng. 98 (2007) 318–327. M. Kotik, A. Archelas, V. Famerova, P. Oubrechtova, V. Kren, J. Biotechnol. 156 (2011) 1–10. D. Hu, C.D. Tang, B. Yang, J.C. Liu, T. Yu, C. Deng, M.C. Wu, J. Ind. Microbiol. Biotechnol. 42 (2015) 671–680. H.B. Tang, V. Krishnakumar, S. Bidwell, B. Rosen, A. Chan, S.G. Zhou, L. Gentzbittel, K.L. Childs, M. Yandell, H. Gundlach, K.F.X. Mayer, D.C. Schwartz, C.D. Town, BMC Genomics 15 (2014) 312. M. Kotik, V. Stepanek, M. Grulich, P. Kyslik, A. Archelas, J. Mol. Catal. B Enzym. 65 (2010) 41–48. W. Xu, J.H. Xu, J. Pan, Q. Gu, X.Y. Wu, Org. Lett. 8 (2006) 1737–1740. M. Kotik, W. Zhao, G. Iacazio, A. Archelas, J. Mol. Catal. B Enzym. 91 (2013) 44–51.
Contents lists available at ScienceDirect
Catalysis Communications journal homepage: www.elsevier.com/locate/catcom
Short communication
Directed modification of a novel epoxide hydrolase from Phaseolus vulgaris to improve its enantioconvergence towards styrene epoxides Hui-Hua Ye a,1, Die Hu b,1, Xiao-Ling Shi a, Min-Chen Wu c,⁎, Chao Deng c, Jian-Fang Li d,⁎ a
School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, PR China Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China Wuxi Medical School, Jiangnan University, Wuxi 214122, PR China d School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China b c
a r t i c l e
i n f o
Article history: Received 1 July 2016 Received in revised form 17 August 2016 Accepted 25 August 2016 Available online 26 August 2016 Keywords: Directed modification Epoxide hydrolase Phaseolus vulgaris Enantioconvergence Styrene epoxides
a b s t r a c t To improve the enantioconvergence of an epoxide hydrolase from Phaseolus vulgaris (PvEH1) towards styrene epoxides, its directed modification was performed based on the rational design by using the molecule docking simulation and the multiple alignment. The single- and multi-site mutation genes of pveh1 were constructed as designed theoretically by PCR, and expressed in E. coli BL21(DE3), respectively. Among all PvEH1 mutants tested, a three-site mutant, PvEH1L105I/M160A/M175I, was selected having the highest activity of 10.66 U/g wet cell and the best regioselectivity (αS N 99%, βR = 86.4%), by which rac-1a was transformed into (R)-1b with 87.8% enantiomeric excess (eep), much higher than that (33.6% eep) by PvEH1. Furthermore, it completely hydrolyzed rac2a–5a into (R)-2b–5b with 52.3–70.9% eep. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Epoxide hydrolases (EHs) can catalyze the kinetic resolution or enantioconvergent hydrolysis of epoxides, retaining epoxide enantiomers or convergently transforming into diols. The products are versatile chiral synthons for the synthesis of highly value-added pharmaceuticals, agrochemicals and fine chemicals [1,2]. The resolution has an intrinsic limitation: the maximum yield of any enantiomer never can exceed 50% [3]. Comparatively, the enantioconvergent hydrolysis, by enzymatic or chemoenzymatic catalysis, can afford diols with high eep and theoretical yield of 100% [4]. The enantioconvergent hydrolysis of epoxides by single EH is an ideal process, but few naturally occurring EHs possess the opposite regioselectivity towards (S)- and (R)-enantiomers [5]. As to plant EHs, only ones from Solanum tuberosum [6] and Vigna radiata [7,8] exhibited good enantioconvergence. Owing to the shortage of enantioconvergent EHs and their advantages in preparing chiral diols, it is desirable to explore more EHs. On the other hand, the enantioconvergent modification of EHs has been performed over the last decade [9]. For example, after five rounds of iterative saturation mutagenesis on Aspergillus niger M200 EH, the best mutant was selected, by which racemic styrene oxide (rac-SO) and para-chlorostyrene oxide (pCSO) were transformed into (R)-diols with over 70% eep [10]. The ⁎ Corresponding authors. E-mail addresses: [email protected] (M.-C. Wu), [email protected] (J.-F. Li). 1 Hui-Hua Ye and Die Hu, the two first authors, contributed equally to this work.
http://dx.doi.org/10.1016/j.catcom.2016.08.036 1566-7367/© 2016 Elsevier B.V. All rights reserved.
exploration and modification of EHs promoted the understanding on the mechanism of enantioconvergent hydrolysis [11]. Recently, a PvEH1-encoding gene (pveh1, GenBank: KR604729) was cloned from P. vulgaris and expressed in E. coli BL21(DE3) in our lab. The enantioconvergent hydrolysis of styrene epoxides by PvEH1 was carried out (Fig. 1). However, the regioselectivity coefficients (αS = 91.1%, βR = 53.3%) towards (S)- and (R)-1a was insufficient, resulting in a low purity of (R)-1b (only 33.6% eep). In this work, to enhance the βR and activity of PvEH1, nine mutant genes were constructed as designed theoretically by PCR and expressed in E. coli BL21, respectively. Using the whole cells of E. coli transformants as catalysts, the properties of PvEH1 and its mutants were characterized and compared.
2. Experimental 2.1. Materials The recombinant plasmid, pET-28a-pveh1, was constructed and preserved in our lab. PrimeSTAR HS DNA polymerase and endonuclease Dpn I (TaKaRa, Dalian, China) were applied for the construction of pveh1 mutants. E. coli BL21(DE3) (Novagen, Madison, WI) was used for the expression of EHs. Rac-SO (1a), (S)- and (R)-1a (Energy, Shanghai, China) were used for the enantioconvergence screening, while rac1a derivatives (2a–5a) (Fig. 1), synthesized by our lab, for the substrate spectrum assay.
H.-H. Ye et al. / Catalysis Communications 87 (2016) 32–35
33
Fig. 1. The enantioconvergent hydrolysis of styrene epoxides into (R)-diols by PvEH1.
2.2. MD simulation and multiple alignment
2.5. Catalytic properties of EHs
Based on the crystal structure of an S. tuberosum EH (PDB: 2CJP), the three-dimensional (3-D) structure of PvEH1 was modeled by MODELLER 9.11 program (http://salilab.org/modeller/). Meanwhile, the 3-D structure of (R)-1a was handled using a ChemDraw Ultra 12.0 software (www.cambridgesoft.com/). The interaction of PvEH1 with (R)-1a, towards whom the βR of PvEH1 is lower, was predicted by MD simulation by AutoDock 4.2 program (http://autodock.scripps.edu) to locate the most suitable binding position and orientation. According to the conformation of PvEH1 docking with (R)-1a, residues in proximity to (R)-1a within 8 Å were confirmed by PyMol software (http://pymol.org/). Plant EHs were searched at the NCBI website (http://blast.ncbi.nlm. nih.gov/) by BLAST server. Then, the multiple alignment of PvEH1 with three EHs having excellent enantioconvergence was performed by ClustalW2 program (http://www.ebi.ac.uk/Tools/msa/). According to the properties of non-conserved residues in proximity to (R)-1a within 8 Å and their positions on PvEH1 3-D structure, several residues were selected to be replaced with the corresponding and frequently emerging ones of S. tuberosum and V. radiata EHs, forming a series of PvEH1 mutants.
The enantioconvergence of EH towards rac-1a was assayed by incubating 475 μL cell suspension and 25 μL rac-1a (10 mM) at 25 °C. After complete hydrolysis, the product was extracted with 1 mL ethyl acetate having 1 mM n-hexanol (internal standard) and analyzed by GC (Table S2). Using (R)- or (S)-1a instead of rac-1a, the regioselectivity coefficients of mutants with enhanced eep were measured. In addition, the substrate spectrum of the best mutant was assayed by replacing rac1a with rac-2a–5a, respectively, by HPLC (Table S2).
2.3. Construction of E. coli transformants The site-directed mutagenesis of pveh1 was conducted by whole-plasmid PCR technique. PCR primers were designed (Table S1). The single-site mutation genes were amplified from pET-28a-pveh1 as following conditions: 30 cycles at 98 °C for 10 s, 55 °C for 15 s and 72 °C for 8 min. The PCR products were digested by DpnI and transformed into E. coli BL21, respectively, forming five E. coli transformants. Similarly, double-site mutation genes, pveh1L105I/M160A and pveh1L105I/M175I, from pET-28a-pveh1L105I were used to construct E. coli transformants, E. coli/pveh1L105I/M160A and E. coli/pveh1L105I/M175I, while pveh1M160A/M175I and pveh1L105I/M160A/M175I separately from pET-28a-pveh1M160A and pET-28a-pveh1L105I/M160A were applied to construct E. coli/pveh1M160A/M175I and E. coli/pveh1L105I/M160A/M175I.
3. Results and discussion 3.1. Selection of mutation sites The highest primary structure identities, analyzed by BLAST server, of PvEH1 with EHs from V. radiata [7], Medicago truncatula [12] and Glycine max (XP_003554633) are 85.7, 81.1 and 81.0%, indicating that PvEH1 was a novel EH. It belongs to the α/β-hydrolase superfamily having two characteristic regions, α/β-hydrolase and lid domains. Its catalytic triad is D101-H299-D264 and two proton donors in the lid are Y150 and Y234 (Fig. S1). According to the docked conformation of PvEH1 with (R)-1a (Fig. 2a), 50 amino acids of PvEH1 in proximity to (R)-1a within 8 Å were located by PyMol software. Based on the multiple alignment of PvEH1 with three plant EHs (VrEH1, VrEH2 and StEH) having excellent enantioconvergence (Fig. 2c), 28 absolutely conserved residues and 14 relatively conserved ones (the residues of PvEH1 were the same as those of two in other three EHs) were excluded. Thus, the remaining eight non-conserved residues were used as the mutant candidates, from which five non-conserved ones, L105, M129, M160, M175 and H267, were selected based on their positions on PvEH1 3-D structure (Fig. 2b) and their distances to catalytic residues and substrate (R)-1a (Table S3). As shown in Fig. 2b, the five selected residues are located near the active center and around (R)-1a. 3.2. Enantioconvergence of EHs towards rac-1a
2.4. Enzyme activity assay After E. coli transformant was induced by 0.5 mM IPTG at 20 °C for 10 h, cells were suspended in 100 mM Na2HPO4-NaH2PO4 buffer (pH 7.0). Cell suspension (1 g wet cell per 10 mL) was used as a catalyst. EH activity was analyzed by HPLC. In brief, 450 μL cell suspension was mixed with 50 μL rac-1a (final concentration of 10 mM). After incubation at 25 °C for 10 min, the reaction was stopped by adding 1 mL methanol. The sample was analyzed using a Waters e2695 system (Waters, Milford, MA) equipped with a C18 column. The mobile phase of methanol/H2O (7:3, v/v) was used at a flow rate of 0.8 mL/min, and monitored at 220 nm using a Waters 2489 UV–Vis detector. One unit (U) of EH activity was defined as the amount of enzyme hydrolyzing 1 μmol rac-1a per minute.
The eep, enantiomeric ratio (E) value and activity of EH towards rac-1a were measured (Table 1). The single-site mutants were constructed by separately replacing residues (L105, M129, M160, M175 and H267) of PvEH1 with the frequently emerging ones (I, L, A, I and Y) of VrEH1, VrEH2 and StEH. The eep values by PvEH1L105I, PvEH1M160A and PvEH1M175I increased by 128, 46 and 58%, respectively, over that (33.6% eep) by PvEH1. In addition, their activities were 3.03-, 1.46- and 4.26-fold higher than that (1.61 U/g wet cell) of PvEH1. As shown in Fig. S2, the expression levels of PvEH1 and its mutants displayed no difference, indicating that the activity improvement of three mutants was not expression amount increase. Furthermore, PvEH1 and PvEH1L105I were isolated from E. coli cells and purified. The specific activity of purified PvEH1L105I was 6.49 U/mg protein, which was 2.82-fold higher than that (2.30 U/mg protein) of purified PvEH1 close to 3.03 folds calculated based on E. coli whole cells. Then,
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Fig. 2. (a) The interaction of PvEH1 with (R)-1a predicted by MD simulation. The catalytic triad of PvEH1 is D101-H299-D264 and two proton donors are Y150 and Y234. (b) Five selected residues, L105, M129, M160, M175 and H267, are located near the active center and around (R)-1a. (c) Multiple alignment of PvEH1 with VrEH1, VrEH2 and StEH. Fifty residues of PvEH1 in proximity to (R)-1a within 8 Å are marked with stars.
according to the different assembly of three mutation sites, four multi-site mutants were constructed. As expected, eep values by PvEH1L105I/M160A, PvEH1L105I/M175I and PvEH1L105I/M160A/M175I were 81.0, 84.1 and 87.8%, while their activities were 4.83, 8.87 and 10.66 U/g wet cell. All eep values Table 1 Catalytic properties of PvEH1 and its mutants towards rac-1a. Epoxide hydrolase
eepa (%)
E valueb
Relative activity (%)
PvEH1 L105I M129 L M160 A M175I H267Y L105I/M160 A L105I/M175I M160 A/M175I L105I/M160 A/M175I
33.6 ± 1.3 76.5 ± 3.2 39.9 ± 1.5 49.2 ± 2.0 53.2 ± 2.5 – 81.0 ± 3.6 84.1 ± 4.1 56.9 ± 2.7 87.8 ± 4.4
1.5 ± 0.1 3.2 ± 0.3 2.1 ± 0.2 2.0 ± 0.1 3.0 ± 0.1 – 2.2 ± 0.1 2.0 ± 0.2 1.8 ± 0.1 3.6 ± 0.2
100 303 ± 14 94 ± 6 146 ± 9 426 ± 17 50 ± 4 300 ± 10 551 ± 26 257 ± 11 662 ± 33
of (R)-1b and (S)-1b. a eep = [(Rp − Sp)/(Rp + Sp)] × 100%. Rp and Sp are the final concentrations. b E = ln (R/R0)/ln (S/S0). R and S are the final concentrations of (R)-1a and (S)-1a, while R0 and S0 the initial concentrations.
by three mutants were higher than those (70.1 and 77.0% eep) by AnEHH:12-A1 and Kau2 [10,13]. 3.3. Regioselectivity coefficients of EHs The regioselectivity coefficients of PvEH1 and its four mutants towards (S)- and (R)-1a were assayed (Fig.3). The βR values of PvEH1L105I, PvEH1L105I/M160A, PvEH1L105I/M175I and PvEH1L105I/M160A/M175I were enhanced from 53.3% to 77.8, 83.0, 84.4 and 86.4%, respectively, while the αS values of four mutants exceeded 99%. The regioselectivity of PvEH1L105I/M160A/M175I was higher than that (αS = 83%, βR = 68%) of mbEH A (renamed VrEH1) [14], but still lower than that (αS = 98%, βR = 93%) of StEH [6]. Usually, the increase of one coefficient would accompany with decrease of the other one, such as the directed evolution of AnEH [10] and Kau2 [13,15]. To our knowledge, it was rare that both αS and βR were synchronously enhanced as reported here. 3.4. Substrate spectrum of PvEH1L105I/M160A/M175I The substrate spectrum of PvEH1L105I/M160A/M175I was assayed (Table 2). The eep values of (R)-2b and 4b catalyzed by the best mutant were
H.-H. Ye et al. / Catalysis Communications 87 (2016) 32–35
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Analysis of the single-site mutants verified that three residue sites, L105, M160 and M175, have notable effects on the eep and activity of PvEH1 towards rac-1a. Then, the four multi-site mutants were constructed according to the different combination of mutation sites. Among all PvEH1 mutants tested, PvEH1L105I/M160A/M175I has the best regioselectivity and highest activity, by which rac-1a was transformed into (R)-1b with high enantiomeric purity. In addition, it showed a broader substrate spectrum. Our present work laid a solid foundation for the modification of this type of EHs to improve their enantioconvergence and activities. Acknowledgment
Fig. 3. The regioselectivity coefficients of a wild-type PvEH1 and its four mutants, PvEH1L105I, PvEH1L105I/M160A, PvEH1L105I/M175I and PvEH1L105I/M160A/M175I, towards (S)and (R)-1a.
increased from 50.3 and 51.4% to 64.7 and 70.9%, respectively. Additionally, PvEH1 L105I/M160A/M175I catalyzed the hydrolysis of rac-3a and 5a into (R)-3b and 5b, respectively, with 52.3 and 69.7% ee p , much higher than those (14.7 and 1.0% eep) by PvEH1. The activity of PvEH1L105I/M160A/M175I was approximately equal to that of PvEH1 towards the same substrate (2a–5a). However, both of them had higher activities towards chloro-substituted styrene epoxides than those towards nitro-substituted ones.
4. Conclusions Based on the rational design, the modification of PvEH1 was performed, which have improved its properties towards styrene epoxides.
Table 2 The substrate spectra and activities of PvEH1 and PvEH1L105I/M160A/M175I. Substrate
1a 2a 3a 4a 5a
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.catcom.2016.08.036. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
PvEH1L105I/M160A/M175I
PvEH1
This work was financially supported by the National Nature Science Foundation of China (No. 21676117), the Fundamental Research Funds for the Central Universities of China (No. JUSRP51412B), and the Postgraduate Innovation Training Project of Jiangsu, China (Nos. KYLX15_1151 and _1190). We are grateful to Prof. Xianzhang Wu (School of Biotechnology, Jiangnan University) for providing technical assistance.
eep (%)
Activity (U/g)
eep (%)
Activity (U/g)
33.6 ± 1.3 50.3 ± 3.3 14.7 ± 0.7 51.4 ± 2.4 1.0 ± 0.1
1.61 ± 0.08 1.30 ± 0.06 4.26 ± 0.16 7.28 ± 0.27 5.83 ± 0.26
87.8 ± 4.4 64.7 ± 1.4 52.3 ± 2.6 70.9 ± 3.5 69.7 ± 3.3
10.66 ± 0.48 1.74 ± 0.08 4.37 ± 0.17 8.50 ± 0.43 5.16 ± 0.23
[12]
[13] [14] [15]
E.Y. Lee, Biotechnol. Lett. 30 (2008) 1509–1514. M. Wang, T. Si, H.M. Zhao, Bioresour. Technol. 115 (2012) 117–125. W.J. Choi, Appl. Microbiol. Biotechnol. 84 (2009) 239–247. M. Kotik, A. Archelas, R. Wohlgemuth, Curr. Org. Chem. 16 (2012) 451–482. N. Bala, S.S. Chimni, Tetrahedron Asymmetry 21 (2010) 2879–2898. M.I. Monterde, M. Lombard, A. Archelas, A. Cronin, M. Arand, R. Furstoss, Tetrahedron Asymmetry 15 (2004) 2801–2805. Q.Q. Zhu, W.H. He, X.D. Kong, L.Q. Fan, J. Zhao, S.X. Li, J.H. Xu, Appl. Microbiol. Biotechnol. 98 (2014) 207–218. Y.W. Wu, X.D. Kong, Q.Q. Zhu, L.Q. Fan, J.H. Xu, Catal. Commun. 58 (2015) 16–20. E.Y. Lee, M.L. Shuler, Biotechnol. Bioeng. 98 (2007) 318–327. M. Kotik, A. Archelas, V. Famerova, P. Oubrechtova, V. Kren, J. Biotechnol. 156 (2011) 1–10. D. Hu, C.D. Tang, B. Yang, J.C. Liu, T. Yu, C. Deng, M.C. Wu, J. Ind. Microbiol. Biotechnol. 42 (2015) 671–680. H.B. Tang, V. Krishnakumar, S. Bidwell, B. Rosen, A. Chan, S.G. Zhou, L. Gentzbittel, K.L. Childs, M. Yandell, H. Gundlach, K.F.X. Mayer, D.C. Schwartz, C.D. Town, BMC Genomics 15 (2014) 312. M. Kotik, V. Stepanek, M. Grulich, P. Kyslik, A. Archelas, J. Mol. Catal. B Enzym. 65 (2010) 41–48. W. Xu, J.H. Xu, J. Pan, Q. Gu, X.Y. Wu, Org. Lett. 8 (2006) 1737–1740. M. Kotik, W. Zhao, G. Iacazio, A. Archelas, J. Mol. Catal. B Enzym. 91 (2013) 44–51.