Stereoselective Hydrolysis of Epoxides by reVrEH3, a Novel Vigna

Article Cite This: J. Agric. Food Chem. 2017, 65, 9861-9870

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Stereoselective Hydrolysis of Epoxides by reVrEH3, a Novel Vigna radiata Epoxide Hydrolase with High Enantioselectivity or High and Complementary Regioselectivity Die Hu,†,‡,# Cunduo Tang,§,# Chuang Li,⊥ Tingting Kan,⊥ Xiaoling Shi,⊥ Lei Feng,† and Minchen Wu*,† †

Wuxi Medical School, ‡School of Food Science and Technology, and ⊥Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China § Nanyang Provincial Engineering Laboratory of Insect Bio-reactor, Nanyang Normal University, Henan 473061, China S Supporting Information *

ABSTRACT: To provide more options for the stereoselective hydrolysis of epoxides, an epoxide hydrolase (VrEH3) gene from Vigna radiata was cloned and expressed in Escherichia coli. Recombinant VrEH3 displayed the maximum activity at pH 7.0 and 45 °C and high stability at pH 4.5−7.5 and 55 °C. Notably, reVrEH3 exhibited high and complementary regioselectivity toward styrene oxides 1a−3a and high enantioselectivity (E = 48.7) toward o-cresyl glycidyl ether 9a. To elucidate these interesting phenomena, the interactions of the three-dimensional structure between VrEH3 and enantiomers of 1a and 9a were analyzed by molecular docking simulation. Using E. coli/vreh3 whole cells, gram-scale preparations of (R)-1b and (R)-9a were performed by enantioconvergent hydrolysis of 100 mM rac-1a and kinetic resolution of 200 mM rac-9a in the buffer-free water system at 25 °C. These afforded (R)-1b with >99% eep and 78.7% overall yield after recrystallization and (R)-9a with >99% ees, 38.7% overall yield, and 12.7 g/L/h space-time yield. KEYWORDS: Vigna radiata, epoxide hydrolase, enantioselectivity, enantioconvergence, styrene oxides, phenyl glycidyl ethers

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INTRODUCTION Epoxide hydrolases (EHs, EC 3.3.2.x) can catalyze the kinetic resolution or enantioconvergent hydrolysis of epoxides, affording enantiopure epoxides and diols that are versatile synthons for the synthesis of agrochemicals, pharmaceuticals, and fine chemicals.1 Examples are as follows: (R)-phenyl glycol was used as a synthetic precursor for arylalkylamine calcimimetics (R)-(+)-NPS R-568 and NK-1 receptor antagonist,2,3 whereas (R)-p-nitrophenyl glycol, (R)-p-chlorophenyl glycol, and (R)-o-cresyl glycidyl ether were precursors separately for β-adrenergic blocker (R)-Nifenalol,4 neuroprotective agent (R)-Eliprodil or antiviral agent EMI37.1,5,6 and cardiovascular hybrid agent Zatebradine.7 Along with the green wave of global industries, the transformation by EHs as an environmentally friendly manner with high stereoselectivity is deemed to be a very attractive alternative to toxic and substrate-limited chemocatalysis.8 Many EHs have been explored and characterized from plants, micro-organisms, and mammals, among which some EHencoding genes have been cloned, modified, and heterologously expressed. The stereochemical outcome of catalytic reaction was highly dependent on the enantio- and regioselectivity of the given EH−epoxide pair.1 The ideal kinetic resolution by a single EH with high enantioselectivity can simultaneously produce enantiopure epoxide and diol with high yields, close to the maximum value of 50%.9 The resolution of transmethylstyrene oxide by Kau2 afforded (1R,2R)-trans-methylstyrene oxide with >99% ees and 48% yield and (1R,2S)-diol with >99% eep and 45% yield.10 However, the majority of EHs exhibited poor enantio- or regioselectivity, resulting in much lower than 50% theoretical yield of chiral epoxides or very low © 2017 American Chemical Society

eep of diols. Recently, limonene EH (LEH) mutants catalyzed the kinetic resolution of racemic styrene oxides and obviously increased the eep and yield of product (R)- or (S)-phenyl glycol compared with wild-type LEH; among them, eep of (R)-phenyl glycol catalyzed by variant SZ649 increased from 21 to 79% eep.11,12 Comparatively, the enantioconvergent hydrolysis can give diols with high eep and 100% theoretical yield.13 The convergent hydrolysis by a single EH can be considered as an ideal bioprocess, but only a few EHs had the high and opposite regioselectivity toward two enantiomers of epoxide.14 For plant EHs, only a Solanum tuberosum EH (StEH) had the high and complementary regioselectivity toward (R)- and (S)-mchlorostyrene oxides, producing (R)-m-chlorophenyl glycol with 97% eep and 88% yield.15 Additionally, two recombinant EHs from Vigna radiata, VrEH1 and VrEH2, were applied to enantioconvergent hydrolysis of p-nitrostyrene oxide, affording (R)-diol merely with 70 and 82.4% eep, respectively.16,17 It also should be stressed that an EH is not likely to function optimally on all types of epoxides. Moreover, EHs for commercial application have generally been limited to insufficient stability, low stereoselectivity, and poor substrate tolerance. Hence, it is necessary to explore more EHs with expected properties, thus providing more options for the kinetic resolution and enantioconvergent hydrolysis of various epoxides. In this work, the full-length cDNA of vreh3, a gene encoding EH from V. radiata (VrEH3), was amplified from total RNA by Received: Revised: Accepted: Published: 9861

August 15, 2017 October 19, 2017 October 23, 2017 October 23, 2017 DOI: 10.1021/acs.jafc.7b03804 J. Agric. Food Chem. 2017, 65, 9861−9870

Article

Journal of Agricultural and Food Chemistry Scheme 1. Stereoselective Hydrolysis of Racemic Epoxides 1a−9a by E. coli/vreh3 Whole Cells Expressing reVrEH3

sequence was obtained by overlapping the amplified 3′- and 5′-end cDNA fragments. According to the full-length cDNA sequence, a pair of specific primers, VrEH-F1 and VrEH-R1, was designed for amplifying both the ORF and DNA sequences of the coding region of vreh3. In brief, the ORF sequence was amplified from the first-strand cDNA using VrEHF1 and VrEH-R1, whereas the DNA sequence was obtained from the genomic DNA using the same primers. The recombinant plasmid containing the ORF sequence flanked by Nde I and Xho I sites, pUCmT-vreh3, was transformed into E. coli JM109 and confirmed by DNA sequencing. Expression and Purification of reVrEH3. The gene vreh3 excised from pUCm-T-vreh3 by Nde I and Xho I was inserted into pET-28a(+) digested using the same enzymes and transformed into E. coli BL21(DE3), generating a recombinant strain, designated E. coli/ vreh3. After the strain was cultured in LB medium containing 50 μg/ mL kanamycin at 37 °C until OD600 reached 0.6−0.8, 0.05 mM IPTG was added to induce the expression of reVrEH3 at 35 °C for 10 h. The induced E. coli/vreh3 cells were collected, suspended in 50 mM Na2HPO4−NaH2PO4 buffer (pH 7.0) containing 500 mM NaCl, and disrupted by sonication at 0 °C. The expressed reVrEH3 with a 6× His tag at its N-terminus was purified by nickel-nitrilotriacetic acid (NiNTA) affinity chromatography.20 SDS-PAGE was used to analyze the expression and purification of reVrEH3. The protein concentration was determined using the BCA-200 protein assay kit (Pierce, Rockford, IL).21 Enzyme Activity Assay. The biocatalysis was carried out in a 1 mL Na2HPO4−NaH2PO4 buffer (50 mM, pH 7.0) system, containing 20 mM racemic 1a and a certain amount of E. coli/vreh3 suspension or purified reVrEH3 solution. After incubation at 25 °C for 15 min, the reaction was stopped by adding 3 mL of methanol. The sample was assayed by high-performance liquid chromatography (HPLC), using a Waters e2695 apparatus (Waters, Milford, MA) equipped with an XBridge C18 column and a 2489 UV−vis detector. One unit (U) of EH activity was defined as the amount of E. coli wet cells or reVrEH3 generating 1 μmol 1b per minute under the above assay conditions. Enzymatic Properties of Purified reVrEH3. The pH optimum of reVrEH3 was determined under the standard assay conditions, except 20 mM 1a in 50 mM Na2HPO4−citric acid buffer (pH 5.0−7.5) and Tris-HCl buffer (8.0−9.0). To estimate the pH stability, aliquots of reVrEH3 were incubated at 25 °C for 1 h at different pH values (Na2HPO4−citric acid buffer: pH 3.5−7.5 and Tris-HCl buffer: 8.0− 9.0). Its pH stability in this work was defined as a pH region over which the residual reVrEH3 activity retained over 85% of its original activity. The temperature optimum of reVrEH3 was determined, at pH optimum, at temperatures ranging from 20 to 60 °C. For evaluating the thermostability, aliquots of reVrEH3 were incubated at various temperatures (20−60 °C) for 1 h. Its thermostability was defined as the temperature at or below which the residual reVrEH3 activity was more than 85%.

RT-PCR, from which an open reading frame (ORF) was obtained, and then expressed in E. coli BL21(DE3). The recombinant (re) VrEH3 was purified, exhibiting the excellent enzymatic properties. Additionally, the catalytic properties of reVrEH3 and stereoselectivity toward nine racemic epoxides (Scheme 1 and Table 2) were investigated. To elucidate the special enantio- and regioselectivity, the interaction of threedimensional structures between VrEH3 and (R)-1a, (S)-1a, (R)-9a, or (S)-9a was analyzed by molecular docking simulation. Finally, the gram-scale preparations of (R)-1b and (R)-9a at high concentration were performed, for the first time, in the buffer-free water system at room temperature, using E. coli whole cells expressing reVrEH3. The product (R)-1b with high eep and yield was prepared by enantioconvergent hydrolysis of 1a, whereas (R)-9a had high ees and yield by kinetic resolution of 9a. These excellent properties make the easily available reVrEH3 a promising candidate for industrial bioprocesses. Further, VrEH3 with the special stereoselectivity also provides a valuable model for understanding the catalytic mechanism of EHs.

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MATERIALS AND METHODS

Materials. V. radiata purchased from a local supermarket (Wuxi, China) was used for total RNA and genomic DNA extraction. E. coli JM109 and plasmid pUCm-T (Sangon, Shanghai, China) were used for gene cloning and sequencing, while E. coli BL21(DE3) and pET28a(+) (Novagen, Madison, WI) were used for expression of vreh3. All enzymes for gene manipulation were purchased from TaKaRa (Dalian, China). Racemic 1a and 7a were products of TCI (Tokyo, Japan), and 2a−6a, 8a, and 9a were synthesized according to the reported methods.18,19 Enantiomers (S)-1a, (R)-1a, (S)-1b, and (R)-1b were purchased from Energy (Shanghai, China). Cloning of the cDNA and DNA Sequences of vreh3. The multiple sequence alignment of known plant EHs, separately from Glycine max, V. radiata, Phaseolus vulgaris, Nicotiana benthamiana, and Solanum tuberosum, was performed using the ClustalW2 (https:// www.ebi.ac.uk/Tools/msa/clustalw2) and ESPript (http://espript. ibcp.fr/). Based on the two highly conserved peptide segments, MHV/IAEK/LG and S/TWRHQI/M, near the N-termini of aligned EHs, the degenerated primers (F1 and F2) were designed. All primers (except for those provided by kits) were synthesized by Sangon (Shanghai, China) and are listed in Table S1. For amplifying the 3′end cDNA of vreh3, the first-strand cDNA was reversely transcribed from the V. radiata total RNA using the RNA PCR kit (TaKaRa, Dalian, China) and then subjected to two runs of PCR (nested PCR). Similarly, for amplifying the 5′-end cDNA, the first-strand cDNA was reversely transcribed using the 5′-full RACE kit (TaKaRa) and subjected to the nested PCR.20 All PCR products were gel-purified and inserted into pUCm-T for DNA sequencing. The full-length cDNA 9862

DOI: 10.1021/acs.jafc.7b03804 J. Agric. Food Chem. 2017, 65, 9861−9870

Journal of Agricultural and Food Chemistry

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The specific activity (U/mg protein) of 1a catalyzed by reVrEH3 was determined under the standard assay conditions, except with the concentrations of (R)-1a or (S)-1a ranging from 2.5 to 25 mM. The kinetic parameters, Km and Vmax, of reVrEH3 were calculated by nonlinear curve fitting function. Stereoselectivity of reVrEH3. Hydrolytic reactions of racemic 1a−9a were performed as follows: 0.9 mL cell suspension containing 100 mg of E. coli/vreh3 wet cells in Na2HPO4−NaH2PO4 buffer (50 mM, pH 7.0) was separately mixed with 0.1 mL of 1a−9a in methanol, at a final concentration of 10 or 20 mM (Table 2), and incubated at 25 °C. Aliquots of the 25 μL sample were periodically withdrawn, extracted with 1 mL of ethyl acetate, and assayed by chiral gas chromatography (GC) or HPLC (Table S2) to calculate the ees, eep, and conversion ratio (c). The enantiomeric ratio (E), (kcatS/KmS)/(kcatR/KmR), represents a degree of preferential hydrolysis of S- over the R-enantiomer, that is, enantioselectivity. The E value of reVrEH3 toward epoxide was calculated as E = ln[(1 − c) × (1 − ees)]/ln[(1 − c) × (1 + ees)].22 The regioselectivity coefficients, αS and αR, were related to attacks at more hindered benzylic carbons (Cα) of (S)- and (R)-epoxide rings, respectively, and calculated as eep = αS − αR + [ees × (1 − c) × (1 − αS − αR)]/c.23 The absolute configurations of enantiomers of 2a−9a and 2b−9b were established by comparing their retention times with those reported previously.12,13,24−26 Molecular Docking Simulation. The three-dimensional (3-D) structure of VrEH3 was modeled by the MODELER 9.11 program (http://salilab.org/modeller/) based on the crystal structure of StEH (PDB: 2CJP) and then optimized by the GROMACS 4.5 package (http://www.gromacs.org/).27 Meanwhile, the 3-D structures of (R)1a, (S)-1a, (R)-9a, and (S)-9a having the lowest energy were handled using the ChembioOffice 2010 package (http://www.cambridgesoft. com/). The molecular docking of VrEH3 with substrate was simulated using the AutoDock 4.2 program (http://autodock.scripps.edu/) to locate the most suitable binding position and orientation. A moleculedocked complex structure was optimized by the GROMACS 4.5 package. The GRPMOS 96 force field and solutions encapsulated by 1.5 nm SPC/E water were used for molecular dynamics (MD) simulations. The molecule-docked complex structures being fixed and unfixed were successively energy minimized by 500 step steepest descent and 1000 steps conjugate gradient algorithms, respectively. Subsequently, the energy-minimized structure models being fixed and unfixed were subjected to a 100 ps and 2 ns MD simulation processes at a temperature of 300 K. The hydrogen bond length (l1 or l2) between the hydroxyl group of Tyr150 or Tyr232 and O atom of the oxirane ring and the distance (dα or dβ) between the O atom of Asp101 and the α-carbon atom (Cα) or β-carbon atom (Cβ) were determined by PyMol (http://pymol.org/).28 Binding free energy of VrEH3 docking with (R)- or (S)-epoxide, as a correlation to the affinity between them, was calculated by the molecular mechanics Poisson− Boltzmann surface area (MM-PBSA) method.29 Gram-Scale Preparation of (R)-1b by Enantioconvergent Hydrolysis. The enantioconvergent hydrolysis of racemic 1a was carried out at 25 °C in a 100 mL buffer-free water system containing 1.20 g of 1a, giving a final concentration of 100 mM, and 10 g of E. coli/vreh3 wet cells. Aliquots of a 25 μL sample were periodically withdrawn to monitor hydrolytic progress by chiral GC. After 1a was completely hydrolyzed, the reaction mixture was saturated with NaCl and extracted with 30 mL ethyl acetate three times. The ethyl acetate fractions were pooled and evaporated at vacuum, followed by recrystallization with CHCl3, affording highly enantiopure (R)-1b. Gram-Scale Preparation of (R)-9a by Kinetic Resolution. The kinetic resolution of racemic 9a was conducted at 25 °C in a 50 mL buffer-free water system containing 1.64 g of 9a (200 mM) and 1 g of E. coli/vreh3 wet cells. The hydrolytic progress was monitored by chiral HPLC. When the ees of (R)-9a reached >99%, the reaction mixture was extracted with 15 mL of ethyl acetate three times. The ethyl acetate fractions were pooled, evaporated at vacuum, and purified by silica gel column chromatography, affording (R)-9a with high enantiopurity.

RESULTS AND DISCUSSION Analysis of the Primary Structure of VrEH3. The fulllength cDNA sequence of 1500 bp was obtained by overlapping

Figure 1. SDS-PAGE analysis of the expressed and purified reVrEH3 (A) and chiral GC assay of racemic 1a and produced (S)- and (R)-1b by reVrEH3 (B). Lane M: standard proteins. Lane 1: E. coli/pET-28a whole cells. Lane 2: E. coli/vreh3 whole cells. Lane 3: purified reVrEH3.

Figure 2. Effects of pH values (A) and temperatures (B) on the catalytic activity and stability of reVrEH3.

Table 1. Kinetic Parameters of Purified reVrEH3 toward (R)- and (S)-1a substrate

Km (mM)

Vmax (U/mg)

kcat (s−1)

kcat/Km (mM−1 s−1)

(R)-1a (S)-1a

53.15 ± 2.31 6.91 ± 0.32

13.49 ± 0.57 8.12 ± 0.38

8.14 4.90

0.15 0.71

both the 1292 bp 3′-end and 338 bp 5′-end cDNA fragments, which were amplified by RT-PCR from V. radiata total RNA. It contains a 94 bp 5′-untranslated region, a 957 bp ORF encoding 318 aa VrEH3, a 435 bp 3′-untranslated region and a 14 bp polyA tail. Additionally, the 1178 bp DNA fragment of vreh3 was PCR-amplified from genomic DNA. Compared with 9863


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Journal of Agricultural and Food Chemistry Table 2. Catalytic Activity and Stereoselectivity of E. coli/vreh3 Whole Cells toward 1a−5a and 7a−9a

enantioconvergent hydrolysisa

kinetic resolution substrate

concn (mM)

activity (U/g wc)

E

αR (%)

αS (%)

time (h)

c (%)

ees (%) conf.

1a 2a 3a 4a 5a 7a 8a 9a

20 10 20 10 20 20 20 20

5.22 1.89 5.67 0.25 3.15 2.77 0.34 4.80

4.4 4.5 6.8 1.0 1.3 11.4 7.9 48.7

2.8 2.6 0.8 11.0 45.3 7.8 5.7 9.3

97.1 84.0 89.6 74.2 95.8 4.6 2.5 4.1

1.0 1.5 1.5 d

65.1 72.2 67.8

91.6 (R) 86.6 (S) 92.2 (R)

33.4 27.8 31.3

2 16 0.75

67.4 69.7 55.0

>99 (R) >99 (R) >99 (R)

26.2 22.5 45.0

a

b

yield (%)

c

time (h) 7 7 13 12 11 15 20 15

eep (%) conf.b yield (%)c 94.1 (R) 85.2 (R) 88.2 (R) 60.9 (R) 50.3 (R) 4.0 (S) 3.2 (S) 3.4 (S)

97.0 93.1 94.1 80.4 75.1

100% conversion ratio. bConfiguration. cAnalytical yield. dNot determined.

6.0−7.5, over which the pH optimum was 7.0. It was highly stable at pH values ranging from 4.5 to 7.5, retaining more than 85% of its original activity (Figure 2A). Additionally, the temperature optimum of reVrEH3, at pH optimum of 7.0, was 45 °C. After being incubated at 20−60 °C for 1 h, reVrEH3 displayed high stability at 55 °C or below and still retained 63% activity at 60 °C (Figure 2B), whose thermostability was much higher than those of the majority of EHs, such as A. usamii EH2 and A. niger M200 EH.20,35 In particular, the optimal temperature and thermostability of VrEH3 were 15 °C higher than those of VrEH2 with only one amino acid difference in the primary structure, whose sequence information was released later (GenBank accession no. AIJ27456).17 The kinetic parameters of reVrEH3 toward two enantiomers of 1a were determined and summarized in Table 1. The Km toward (S)-1a (KmS) was 6.91 ± 0.32 mM, which was 7.7-fold lower than KmR of 53.15 ± 2.31 mM, indicating that reVrEH3 displayed higher affinity or preferential hydrolysis toward (S)1a. Although kcatS of 4.90 s−1 was lower than kcatR of 8.14 s−1, the catalytic efficiency (kcat/Km) toward (S)-1a (0.71 mM−1 s−1) was much higher than that toward (R)-1a (0.15 mM−1 s−1). The ratio of catalytic efficiencies, (kcatS/KmS)/(kcatR/ KmR), was calculated to be 4.7. All of these results indicated that reVrEH3 possesses a moderate enantioselectivity toward racemic 1a. Analysis of Substrate Spectrum of reVrEH3. To explore the application and catalytic mechanism of reVrEH3 toward different substrates, six styrene oxides 1a−6a and three phenyl glycidyl ethers 7a−9a were selected and subjected to the hydrolytic reactions using E. coli/vreh3 whole cells as the biocatalyst. The catalytic activities of whole cells toward eight epoxides were determined to be 0.25 to 5.67 U/g wet cell (Table 2), but no EH activity was detected toward onitrostyrene oxide 6a, which was identical to mbEHs A and B isolated from mung bean.13 This phenomenon may be attributed to the steric hindrance caused by ortho-substitution. In addition, the hydrolytic progress curves of 1a−5a and 7a−9a catalyzed by reVrEH3 were determined (Figure 3), revealing its different catalytic properties toward various epoxide substrates. Enantioselectivity of reVrEH3 toward 1a−5a and 7a− 9a. Corresponding to the c values of eight epoxides ranging from 30 to 50%, the E values of reVrEH3 were calculated (Table 2) based on the ees values of retained enantiomers of epoxides. In detail, reVrEH3 exhibited no or very low enantioselectivity or preference toward racemic 4a or 5a (E value of 1.0 or 1.3), moderate enantioselectivities toward 1a− 3a and 7a−8a (E values from 4.4 to 11.4), as well as the high enantioselectivity toward 9a (E value of 48.7). The kinetic resolutions of 1a−3a and 7a−9a were carried out at 25 °C by

the full-length cDNA, the coding region DNA has two introns of 142 and 79 bp (Figure S1). Many valuable enzymes have been identified and characterized from micro-organisms, among which some recombinant enzymes have been applied for biotransformation.30−32 It is also an effective approach to explore more biocatalysts from various, easily available and cheap crops.13,33 To date, only a few plant EH genes have been cloned and expressed;1 the successful cloning of vreh3, therefore, enriched the biological information on EHs and their genes. A primary structure of VrEH3 was deduced from ORF of vreh3 (GenBank accession no. KR013755), sharing 57.8 and 72.4% identities separately with those of StEH (AAA81891) and VrEH1 (ADP68585).16,34 A BLAST search showed that VrEH3 shared the highest identity of 91.5% with a hypothetical EH from P. vulgaris (XP_007147004). The multiple sequence alignment of six plant EHs displayed that VrEH3 contains three typical conserved motifs, which are identical to those of an α/β fold EH family: HGXP, GXSmXS/T, and SmXNuXSmSm, where X, Sm, and Nu represent any residue, small residue, and nucleophilic residue, respectively. One catalytic triad (Asp101− Asp262−His297) and two proton donors (Tyr150 and Tyr232) in VrEH3 were predicted (Figure S2). Expression and Purification of reVrEH3. After the recombinant strain, E. coli/vreh3 or E. coli/pET-28a (a negative control strain), was induced by 0.05 mM IPTG at 35 °C for 10 h, the EH activity of E. coli/vreh3 whole cells toward 1a was measured to be 5.22 U/g wet cell (wc), whereas no EH activity was detected in E. coli/pET-28a whole cells. SDS-PAGE analysis displayed that the apparent molecular weight of reVrEH3 fused with an extra 20 aa oligopeptide containing a 6× His tag at its N-terminus was 36.8 kDa, which was consistent with its theoretical one (36507 Da) (Figure 1A). reVrEH3 was purified to homogeneity by Ni-NTA affinity column chromatography. The specific activity of reVrEH3 toward 1a was assayed to be 4.71 U/mg protein, which was higher than those of EHs from Aspergillus niger M200 (0.64 U/ mg),35 Agrobacterium radiobacter AD1 (1.04 U/mg),36 and S. tuberosum (2.0 U/mg).15 Interestingly, reVrEH3 exhibited excellent enantioconvergence toward racemic 1a, affording (R)-1b with 94.1% eep at 100% conversion (Figure 1B and Table 2). To the best of our knowledge, the eep value of (R)-1b from enantioconvergent hydrolysis of 1a by a single reVrEH3 was higher than those by CcEH (90% eep),37 StEH (86% eep),15 Kau2 (77% eep), and Kau8 (71% eep).10 Enzymatic Properties of Purified reVrEH3. The enzymatic properties of purified reVrEH3 in this study were investigated using styrene oxide 1a as the model substrate. The reVrEH3 exhibited higher catalytic activity at a pH range of 9864


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Figure 3. Hydrolysis progress curves of 1a−5a and 7a−9a catalyzed by E. coli/vreh3 whole cells. Hydrolysis reactions of racemic 1a−5a and 7a−9a were performed as follows: 0.9 mL cell suspension containing 100 mg of E. coli/vreh3 wet cells in Na2HPO4−NaH2PO4 buffer (50 mM, pH 7.0) was separately mixed with 0.1 mL of 1a−5a and 7a−9a in methanol, at a final concentration of 10 or 20 mM and incubated at 25 °C. Aliquots of the 25 μL sample were periodically withdrawn and assayed by chiral GC or HPLC to calculate the concentrations of (S)-epoxide (▲) and (R)-epoxide [(R)-1a, 3a−5a are shown in black Δ, (R)-2a in red Δ, and (R)-7a−9a in cyan Δ], ees of epoxide (○), eep of diol (●) and c (◆).

E. coli/vreh3 whole cells, affording five (R)-epoxides with ees values from 86.6 to >99% and yields from 27.4 to 45.0%, respectively, while giving (S)-2a with an ees of 86.6% and yield of 27.8%. However, styrene oxides 4a−5a were not suitable for kinetic resolution by reVrEH3 due to its no or low enantioselectivity. These results indicated that the positions and electronic properties of the substituents of epoxides greatly influence the catalytic activity and stereoselectivity of reVrEH3.

To our knowledge, reVrEH3 displayed the highest enantioselectivity toward 9a for remaining (R)-9a among all known native EHs, such as those from Trichosporon loubierii (E = 41)38 and A. niger LCP (E = 5),28 indicating that it was an ideal biocatalyst for the kinetic resolution of racemic 9a. Regioselectivity of reVrEH3 toward 1a−5a and 7a−9a. The regioselectivity coefficients, αR (or βR = 1 − αR) and αS (or βS = 1 − αS), were applied to interpret the enantioconvergent 9865


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Figure 4. Molecular docking simulations between VrEH3 and (R)-1a (A), (S)-1a (B), (R)-9a (C), or (S)-9a (D). Active site residues including a catalytic triad Asp101−Asp262−His297 and two proton donors Tyr150 and Tyr232 are shown as cyan sticks. The hydrogen bond length (l1 or l2) between the hydroxyl group of Tyr150 or Tyr232 and O atom of the oxirane ring is shown as a green line. The distance (dα or dβ) between the O atom of Asp101 and Cα or Cβ is shown as a black line.

Table 3. Molecular Docking Simulation Parameters of VrEH3 with (R)-1a, (S)-1a, (R)-9a, and (S)-9a l1 (Å)

l2 (Å)

dα (Å)

ΔGbind (kcal mol−1)

dβ (Å)

substrate

R

S

R

S

R

S

R

S

R

S

1a 9a

2.9 3.4

3.2 3.2

3.4 2.8

2.8 2.6

4.4 4.1

2.9 3.1

3.0 3.4

4.0 2.8

−20.8 −6.0

−31.7 −25.3

Figure 5. Regioselectivity of reVrEH3 toward two enantiomers of 1a (A) and 9a (B).

45.3 and αS = 95.8%), but hardly any regioselective complementarities toward 7a−9a (Table 2). For the two enantiomers of 2a, the regioselective complementarity of reVrEH3 was higher than that of VrEH1 (αR = 13 and αS = 83%)16 but close to that of VrEH2 (αR = 1.6 and αS = 87.2%).17 Due to the high and complementary regioselectivity as well as low enantioselectivity, the enantioconvergent hydrolysis of 1a, 2a, or 3a by E. coli/vreh3 whole cells produced (R)-diol with 94.1, 85.2, or 88.2% eep and 97.0, 93.1, or 94.1% yield at 100% conversion. In our previous work, the regioselectivity of PvEH1 toward 1a was improved by directed evolution. Owing to the increases of αS and βR from 91.1 to >99% and 53.3 to 86.4%, respectively, the eep of product (R)-1b from enantioconvergent hydrolysis of 1a by PvEH1L105I/M160A/M175I, a three-site mutant, was obviously increased from 33.6 to 87.8%.14

hydrolysis of racemic epoxides.1 Here, αR and αS values of reVrEH3 toward α-carbon atoms of (R)- and (S)-1a−5a and 7a−9a were calculated (Table 2), respectively, based on the c of epoxides, ees of retained enantiomers, and eep of produced diols in the hydrolytic process. As a comparison, the calculated selectivity coefficients toward 1a (αR = 2.8 and αS = 97.1%) were very consistent with those (αR = 2.5 and αS = 96.7%) directly determined using (R)-1a and (S)-1a as the substrates. The regioselective complementarity, that is, the difference between αS and αR values, of reVrEH3 toward two enantiomers of 1a was higher than those of mbEH A (αR = 32 and αS = 83%)13 and StEH (αR = 7 and αS = 98%).15 In addition, reVrEH3 also possessed the high and complementary regioselectivity toward (R)- and (S)-2a or 3a (αR = 2.6 or 0.8%, and αS = 84.0 or 89.6%) and the lower complementarity toward (R)- and (S)-4a (αR = 11.0 and αS = 74.2%) or 5a (αR = 9866


Article


Molecular Docking Simulation between VrEH3 and Substrate. Molecular docking simulation between VrEH3 and substrate was performed to gain insights into the substrate binding mode and the origin of different enantio- or regioselectivity.43 The (R)-1a, (S)-1a, (R)-9a, or (S)-9a was automatically docked into the SBP of VrEH3 using the AutoDock 4.2 program. Molecular docking models showed that both (R)-1a and (S)-1a docked in the much broader tunnel 1, whereas (R)-9a docked in tunnel 1 and (S)-9a in tunnel 2 (Figure 4). As show in Table 3, the hydrogen bond lengths (l1 or l2) toward both enantiomers of 1a and 9a are in the range of 2.8− 3.4 Å, indicating that the substrate binding and potential activation by two hydrogen bonds from Tyr150 or Tyr232 are maintained, which is one requirement needed to be fulfilled for a smooth oxirane ring-opening reaction.44 As discussed by Bruice, the catalytic efficiency for substrates is dependent on how often the nucleophile and electrophile are present in near attack conformation.45 Here, the distances dα and dβ for (S)-1a and (R)-1a (dα = 2.9 and dβ = 3.0) were much shorter than those for (R)-1a and (S)-1a (dα = 4.4 and dβ = 4.0), indicating that Cα of (S)-1a and Cβ of (R)-1a are nearly attacked by Asp101, thus racemic 1a could be enantioconvergently hydrolyzed by reVrEH3 (Figure 5A). Additionally, (S)-9a has relatively shorter distances (dα = 3.1 and dβ = 2.8) and lower ΔGbind (−25.3 kcal mol−1) than those of (R)-9a, indicating that VrEH3 favors (S)-9a, thus being preferentially hydrolyzed (Figure 5B). Those analytical results suggested that the SBP plays a crucial role in the substrate specificity and stereoselectivity of VrEH3 by influencing the binding and interaction between active site residues and the substrate. Preparation of (R)-1b by Enantioconvergent Hydrolysis Using E. coli/vreh3 Whole Cells. To access the practical capability of reVrEH3, a gram-scale enantioconvergent hydrolysis of racemic 1a (1.20 g, 100 mM) was performed using 10 g of E. coli/vreh3 wet cells in a 100 mL buffer-free water system. The conversion reached nearly 82% within 8 h, after which the reaction rate decreased rapidly, which may be caused by the high Km of residual (R)-1a. After incubation at 25 °C for 22 h, (R)-1b was obtained with 92.5% eep at 100% conversion (Figure 6A). Then, 1.09 g of (R)-1b was obtained with eep up to >99% at 78.8% overall yield after simple recrystallization with CHCl3. Thus far, a few reported EHs were applied to prepare enantiopure diols at high concentration, due to enzyme instability, substrate water dissolubility, or product inhibition during the biotransformation process.1 Chen et al. used the hydrophilic ionic liquids in a two-phase system, improving the substrate concentration of racemic 1a only from 20 mM to 35 mM catalyzed by mung bean with a 49.4% yield of (R)-1b.47 Compared with other EHs in Table 4, the enantioconvergent hydrolysis of racemic 1a can effectively performed by E. coli/verh3 whole cells at a concentration as

Figure 6. Time course curves of the enantioconvergent hydrolysis of racemic 1a (A) and the kinetic resolution of racemic 9a (B).

Homology Modeling of VrEH3 3-D Structure. Based on the crystal structure of StEH (PDB: 2CJP),27 sharing 57.8% primary structure identity with VrEH3, the 3-D structure of VrEH3 was homologically modeled. The VrEH3 3-D structure can be divided into two parts: a α/β-hydrolase domain that contains a catalytic triad Asp101−Asp262−His29 and a lid domain that contains two proton donors Tyr150 and Tyr232 (Figure S3). Interestingly, it contains a “U” shape substrate binding pocket (SBP) with the active site residues placed in the center, giving rise to two narrow tunnels (tunnel 1 and tunnel 2), whose spatial properties have an effect on the substrate preference.28,39 Tunnel 1 was constituted of W102, I105, V126, P127, L129, T137, A143, M144, I151, V264, S267, L268, and M270, whereas tunnel 2 was constituted of F33, P34, I176, T179, K181, G183, P185, N197, M263, and F298 (Figure S3). Both tunnels of VrEH3 SBP are interlinked with each other and also lead eventually to the exterior of the protein, which dramatically differs with those SBPs of other α/β hydrolase fold EHs, such as StEH (PDB: 2CJP),27 AnEH LCP (PDB: 3G0I),40 BmEH (PDB: 4G02),41 and MtEHB (PDB: 2ZJF),42 whose tunnels closed at the internal region.

Table 4. Preparation of (R)-1b by Enantioconvergent Hydrolysis of Racemic 1a Using Several EHs enzyme source

catalytic form

reaction system

substrate concn (mM)

temp (°C)

time (h)

c (%)

eep (%)

C. crescentus S. tuberosum Kau2 mung bean V. radiate

cells re-enzymeb re-enzyme enzyme powder E. coli cells

phosphate buffer phosphate buffer Tris−HCl buffer n-hexane/ionic liquid buffer-free water

10 4 3.5 30 100

25 28 28 35 25

2

93 100 100

22

100

90 (R) 86 (R) 77 (R) 95 (R) 92.5 (R)

a

yield (%) a

49.4 96.2c

ref 37 15 10 47 this study

No information. bRecombinant enzyme. cAnalytical yield. 9867


Article

Journal of Agricultural and Food Chemistry Table 5. Preparation of (R)-9a by Kinetic Resolution of Racemic 9a Using Several Native EHs

a

enzyme source

catalytic form

reaction system

concn (mM)

temp (°C)

time (h)

ees (%)

T. loubierii A. niger LCP B. alcalophilus B. megaterium V. radiate

cells re-enzymeb growing cells re-enzyme E. coli cells

phosphate buffer phosphate buffer culture medium phosphate buffer deionized water

45 10 6 183 200

30 25 30 30 25

6 a 24

>99 (R) − (R) >99 (S) >98 (S) >99 (R)

No information. bRecombinant enzyme.

1

■

high as 100 mM in a single buffer-free water system, which has easy operation, is more environmentally friendly, and benefits the postprocessing. Considering the high concentration, eep, and yield of (R)-1b, the applicability of this enantioconvergent process is also markedly superior to those of other reported EHs. (R)-Phenyl glycol 1b: white solid; 1.09 g; [α]D20 −41.2 (c 0.50, ethanol), >99% ees; 1H NMR (400 MHz, CDCl3, TMS) δ 7.36−7.26 (m, 5H), 4.95 (dd, J1 = 8.0 Hz, J2 = 3.6 Hz, 1H), 3.85 (dd, J1 = 11.2, J2 = 3.6 Hz, 1H), 3.64 (dd, J1 = 11.2 Hz, J2 = 8.0 Hz, 1H). Preparation of (R)-9a by Kinetic Resolution Using E. coli/vreh3 Whole Cells. A gram-scale kinetic resolution of racemic 9a (1.64 g, 200 mM) was performed using 1 g of E. coli/vreh3 wet cells in a 50 mL buffer-free water system. (R)-9a with >99% ees and 44% yield and product (S)-9b with 76.2% eep and 42% yield were obtained after incubation for 1 h at 25 °C (Figure 6B). After being purified by silica gel column chromatography, 0.64 g of (R)-9a was obtained in >99% ees at 38.7% overall yield, as calculated on the basis of the racemic 9a, which accounts for up to 77.4% of the theoretical yield. To our knowledge, some reported EHs favor (R)-9a possessing high enantioselectivity, such as those from Bacillus alcalophilus24 and B. megaterium,46 and only a few EHs could preferentially hydrolyze (S)-9a while retaining the useful (R)-9a (Table 5). In addition, although Tsukamurella paurometabola EH exhibited excellent activity and enantioselectivity (E = 58) toward 7a, no activity was detected for 9a.48 In our work, the kinetic resolution showed the highest substrate concentration and space-time yield (200 mM, 12.7 g/L/h) for the biocatalytic synthesis of (R)-9a catalyzed by reported EHs, which were 4.5and 25.9-fold higher than those (45 mM, 0.49 g/L/h) achieved with T. loubierii.38 Further studies on optimizing reVrEH3 expression and biotransformation processes are in progress to enhance the utility of reVrEH3 for industrial application. (R)-oCresyl glycidyl ether 9a: colorless oil; 0.64 g; [α]D20 −13.56 (c 0.50, methanol), >99% ees; 1H NMR (400 MHz, CDCl3, TMS) δ 7.13−7.16 (m, 2H), 6.9 (t, J = 7.6 Hz, 1H), 6.8 (d, J = 8.0 Hz, 1H), 4.25 (dd, J1 = 3.2 Hz, J2 = 11.2 Hz, 1H), 4.00 (q, J = 5.2 Hz, 1H), 3.36−3.40 (m, 1H), 2.92 (t, J = 4.4 Hz, 1H), 2.8 (dd, J1 = 2.8 Hz, J2 = 4.8 Hz, 1H), 2.25 (s, 3H). In conclusion, a new plant EH gene from V. radiate was successfully cloned and expressed in E. coli BL21(DE3). The reVrEH3 displayed high specific activity, excellent thermostability, and different stereoselectivity toward a broad range of epoxides. Interestingly, reVrEH3 exhibited very high and complementary regioselectivity and low enantioselectivity toward 1a−3a, which is suitable for their enantioconvergent hydrolysis. Meanwhile, it also had high enantioselectivity but no complementary regioselectivity toward 9a, which is suitable for its kinetic resolution. Gram-scale preparations of (R)-1b and (R)-9a with high enantiopurity (>99 ee), tolerance to high substrate concentration, and high yield make reVrEH3 an attractive biocatalyst for industrial bioprocesses.

yield (%)

STY (g/L/h)

ref

40

0.49

38 32 38.7

0.015

38 28 24 46 this study

12.7

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b03804. Primers for PCR amplification in this study (Table S1); chiral GC and HPLC conditions for analysis of epoxides and diols (Table S2); nucleotide sequence of vreh3 and its deduced amino acid sequence of VrEH3 (Figure S1); multiple alignment of six plant EH primary structures (Figure S2); 3-D structure of VrEH3 (A) and the surface view of its substrate binding pocket (B) (Figure S3); chiral GC/HPLC spectra used to determine the configuration of epoxides 1a−9a and diols 1b−9b (Figures S4−S6) (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Fax: +86 510 85327332. E-mail: [email protected]. ORCID

Minchen Wu: 0000-0003-2833-8436 Author Contributions #

D.H. and C.T. contributed equally to this work as a first author.

Funding

This work was financially supported by the National Natural Science Foundation of China (21676117), the Postgraduate Innovation Training Project of Jiangsu, China (KYLX16_0804 and SJLX16_0472). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We are grateful to Prof. Xianzhang Wu (School of Biotechnology, Jiangnan University) for providing technical assistance.

■

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Stereoselective Hydrolysis of Epoxides by reVrEH3, a Novel Vigna

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