Directed evolution of metagenome-derived epoxide hydrolase for improved enantioselectivity and enantioconvergence

Journal of Molecular Catalysis B: Enzymatic 91 (2013) 44–51

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Directed evolution of metagenome-derived epoxide hydrolase for improved enantioselectivity and enantioconvergence Michael Kotik a,∗ , Wei Zhao b , Gilles Iacazio b , Alain Archelas b a b

Laboratory of Biotransformation, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4, Czech Republic ˇ Université d’Aix-Marseille, CNRS, iSm2 UMR 7313, Avenue Escadrille Normandie Niemen, 13397 Marseille, France

a r t i c l e

i n f o

Article history: Received 26 November 2012 Received in revised form 25 February 2013 Accepted 25 February 2013 Available online 5 March 2013 Keywords: Regioselectivity Enantioselectivity Chiral building block Deracemization para-Chlorostyrene oxide

a b s t r a c t We performed a directed evolution study with a metagenome-derived epoxide hydrolase (EH), termed Kau2. Homology models of Kau2 were built; we selected one of them and used it as a guide for saturation mutagenesis experiments targeted at specific residues within the large substrate binding pocket. During the molecular evolution process, we found several enzyme variants with higher enantioselectivity or enhanced enantioconvergence toward para-Chlorostyrene oxide. Improved enantioselectivities by up to a factor of 5, reaching an E-value of up to 130 with the R-enantiomer as the residual epoxide, were achieved by replacing amino acid pairs at the positions 110 and 113, or 290 and 291, which are positions located in the vicinity of two presumed binding sites for the epoxide enantiomers. The (R)-paraChlorophenylethane-1,2-diol product exhibited a high enantiomeric excess (ee) of 97% at 50% conversion of the racemic epoxide for the most enantioselective variant. Further, five amino acid substitutions were sufficient to substantially increase the degree of enantioconvergence and to lower the E-value to 17 for the final evolved EH variant, enabling the production of the R-diol with an ee-value of 93% at 28 ◦ C in a complete conversion of the racemic epoxide. Higher eep -values of up to 97% were determined in enantioconvergent reactions using lower temperatures. The EH activities of whole cells were found to be within the range of 74–125% of the wild-type activity for all investigated variants. We show in this report that the metagenome-derived Kau2 EH is amenable to the redesign of its enantioselectivity and regioselectivity properties by directed evolution using a homology model as a guide. The generated enzyme variants should be useful for the production of the chiral building blocks (R)-para-Chlorostyrene oxide and (R)-para-Chlorophenylethane-1,2-diol. © 2013 Elsevier B.V. All rights reserved.

1. Introduction It is now established that the use of biocatalysts can be an attractive alternative to a purely chemical synthesis approach. As a result, enzymes are increasingly being used as biocatalysts in the chemical and pharmaceutical industry, predominantly for the synthesis of enantiomerically pure compounds [1]. The demand for enzymes with novel substrate specificities and high stereospecificities is also growing rapidly due to legislational, environmental and economic issues. It appears, however, that many wild-type enzymes do not perfectly fulfill the requirements for a highly active and stereospecific catalyst for the synthesis of a particular pharmaceutical building block or precursor [2]. The protein engineering of existing biocatalysts and the discovery of novel enzymes are important strategies for overcoming this bottleneck. The number of uncharacterized enzymes is enormous given the high degree of microbial

∗ Corresponding author. Tel.: +420 241062569; fax: +420 241062509. E-mail address: [email protected] (M. Kotik). 1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molcatb.2013.02.006

biodiversity in microcosms such as soil or groundwater, which is much larger than previously anticipated [3,4]. This rich source of genes which encode as-yet uncharacterized enzymes is slowly being uncovered. The number of solved X-ray protein structures has now reached a substantial level, which often enables us to build useful structural models of novel target enzymes using homology modeling algorithms [5]. This modeling approach is based on an existing homologous protein whose crystal structure has been solved. The generated homology model can then be used as a guide for saturation mutagenesis at selected sites aimed at improving the target enzyme using directed evolution. This directed evolution study focused on an epoxide hydrolase (EH) whose gene was recently retrieved from the genomic DNA of a microbial biofilter community [6]. In a previous study, the enzyme was characterized and assessed for its usefulness as a biocatalyst in preparative-scale resolutions and enantioconvergent biotransformation reactions. It turned out that this EH, denoted Kau2, has a broad substrate specificity with a considerable degree of enantioconvergence [6]. The high activity of this enzyme with

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Scheme 1. Hydrolysis of rac-1 using wild-type Kau2 EH: attack at the oxirane ring by the catalytic nucleophile Asp-109 occurs at carbon atom C1 (␣-attack), which leads to the formation of 2 with an inverted configuration by following the ˛R or ˛S pathway. Attack also occurs at carbon atom C2 (ˇ-attack), resulting in 2 with an retained configuration (following the ˇR or ˇS pathway). The composition of the enantiomers of 2 after complete conversion of the racemic epoxide gives an eep -value of 84%. The percentage of epoxide molecules following a particular pathway depends on the regioselectivity coefficients ˛R , ˛S , ˇR , and ˇS ; their values were determined for the wild-type Kau2 EH to be: ˛R = 11.0%, ˛S = 95.3%, ˇR = 89.0%, and ˇS = 4.7%.

para-Chlorostyrene oxide (1) prompted us to improve this biocatalyst further to higher degrees of enantioselectivity or enantioconvergence. The screening methodology applied in this report enabled the selection of EH variants with improved enantioconvergence and potentially higher enantioselectivity, which are both valuable characteristics of EHs used in biotransformations. Enantioselective EHs are applied in hydrolytic kinetic resolutions and lead to the formation of enantiopure epoxides, which are used as chiral synthons and in click chemistry [7–10]. Enantioconvergent EHs enable the production of enantiopure vicinal diols, another class of chiral building blocks [11–13], with a theoretical yield of 100% [14]. Further, vicinal diols can very often be chemically transformed into the corresponding epoxides with no loss in enantiomeric excess (ee). Moreover, 1,2-diols can be converted into cyclic sulfites and sulfates, which are another valuable class of reactive epoxide-like synthons [12]. The main diol product of Kau2 with rac-1 as the substrate is (R)-para-Chlorophenylethane-1,2diol (2), which is for instance a key building block in the synthesis of Eliprodil, a neuroprotective agent [15]. A strong motivation for our laboratory evolution study can be found in the lack of synthetic catalysts which would enable the enantioconvergent production of vicinal diols from racemic epoxides in a deracemization reaction. Wild-type EHs with high degrees of enantioconvergence are not frequent, although occasionally high ee-values for the generated diols have been reported [14]. Besides high biocatalyst activity, the following EH characteristics are desirable from a practical point of view: (1) high enantioselectivity with ideally a high proportion of either ˛- or ˇ-attack at the oxirane ring of the fast enantiomer, resulting in a high eep (see below and Scheme 1), and (2) high enantioconvergence in conjunction with low enantioselectivity, since high enantioselectivity would only prolong the reaction time to reach 100% conversion of the epoxide substrate. Lacking an X-ray structure, we constructed a homology model of Kau2 that allowed us to target sites within the substrate binding cavity for saturation mutagenesis experiments. We performed eight rounds of library creation and selection in total, resulting in a small collection of variants that fulfill to a high degree the abovementioned properties of perfect EHs.

2. Materials and methods 2.1. Chemicals and reagents 4-(4-nitrobenzyl)pyridine, ethanolamine, Tween 80, and rac-1 were purchased from Sigma–Aldrich. Epoxycyclooctane was supplied by Merck. The growth media components were from Oxoid

Ltd. The inhibitor compounds N-cyclohexyl-N -decylurea (CDU) and N-cyclohexyl-N -(4-iodophenyl)urea (CIU) were synthesized as described in Argiriadi et al. [16]. (R)-1, (S)-1, (R)-2, and (S)-2 were synthesized according to Schaus et al. [17] with ee-values of 99, 99, 96, and 95%, respectively. 2.2. Library construction and iterative saturation mutagenesis Libraries were constructed by PCR using either the Phusion site-directed mutagenesis kit (Finnzymes Oy) or the QuickChange Lightning site-directed mutagenesis kit (Agilent Technologies) according to the manufacturers’ instructions. The primers used for saturation mutagenesis are shown in Table S1 of the Supplementary material. The pSEKau2 plasmid, which harbors the EH-encoding metagenomic DNA [6], was used as the template. Escherichia coli BL21-Gold cells (Agilent Technologies) were transformed with the mutagenized plasmid, and the resulting colonies were transferred into 96-well plates as described in Kotik et al. [18]. We selected M. Reetz’s iterative saturation mutagenesis approach as our directed evolution strategy [19]. 2.3. EH inhibition assay Kau2 EH activities were determined by HPLC in the presence of (S)-1 (0.5 − 4.0 mM) and various inhibitor concentrations (0 − 360 nM), monitoring the diol product formation at 220 nm using a Nucleosil C18 column (250 mm × 4.6 mm, i.d. 5 ␮m, Macherey Nagel). An isocratic mixture of 55% acetonitrile and 45% water (vol.) was applied for diol elution at a flow-rate of 0.5 mL min−1 (tr : 6.2 min). The injection volume was 20 ␮L. The type of inhibition and the inhibition constant Ki were determined according to Cortes et al. [20]. 2.4. Screening for activity and enantioconvergence The colorimetric activity screening was performed with whole cells in the presence of 6.6 mM rac-1 using 96-well plates as described in a previous report [18]. Each active clone was reanalyzed for the degree of enantioconvergence in two separate reactions using a cell suspension and either enantiomer of 1 as the substrate: one half of the cell suspension was incubated for 30 min with 3.7 mM (S)-1, the other half was incubated for 90 min with 3.7 mM (R)-1 at 28 ◦ C. The ee of 2, which was formed in each separate reaction from either (R)-1 or (S)-1, was determined by chiral GC using a CycloSil-B column (30 m, 0.25 mm diameter, Agilent Technologies). The internal standard epoxycyclooctane, 1, (S)-2, and (R)-2 eluted at an isothermal oven temperature of 185 ◦ C at

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1.8, 2.2, 7.9, and 8.1 min, respectively, using H2 as the carrier gas (see Fig. S1 of the Supplementary material). The determined ee of 2 enabled rapid calculation of the regioselectivity coefficients ˛S and ˛R and the degree of enantioconvergence, as described elsewhere [18]. 2.5. Determination of enantioselectivities and regioselectivity coefficients Cultivations of selected E. coli clones in LB medium and EH overexpression were performed as described elsewhere [18]. Whole cells were used for the biotransformation experiments. E-values were determined in kinetic resolution experiments following ees and the extent of conversion c on an 8-mL scale in buffer A (50 mM phosphate buffer, 1 mM EDTA, and 2 mM 2-mercaptoethanol. pH 6.8) with 3.9 mM rac-1 and 8% ethanol at 28 ◦ C. Chiral GC analysis of 1 was performed on a CycloSil-B capillary column at an isothermal temperature of 120 ◦ C: epoxycyclooctane (internal standard), (R)-1, and (S)-1 eluted at 8.0, 14.1, and 14.9 min, respectively. The regioselectivity coefficients ˛S and ˛R were determined in biotransformation reactions at 28 ◦ C with rac-1, following c, ees and eep . After extracting the remaining epoxide with iso-octane, the diol 2 formed was extracted with 2,2-dimethoxypropane and derivatized to its acetonide in the presence of Amberlite IR-120 resin. The ees of the acetonides were determined by chiral GC analysis using a CP-Chirasil-Dex CB capillary column (Agilent Technologies) at an isothermal temperature of 135 ◦ C with the following retention times: (R)-2a, 10.5 min, and (S)-2a, 11.6 min. Plotting eep against ees ·(1 – c)·c−1 enabled the determination of ␣R and subsequently ␣S as described in Kotik et al. [6], using non-absolute values for eep and ees according to Moussou et al. [21] (see Figs. S2 and S7 of the Supplementary material). 2.6. Phylogenetic trees and molecular modeling simulations Phylogenetic trees were constructed with the Neighbor-Joining method using the software MEGA 4.0 [22]. Structural homology models of Kau2 were generated using SWISS-MODEL, an automated modeling server, using the default settings [5]. Computational docking experiments were performed in AutoDock 4.0 using the Lamarckian genetic algorithm of AutoDockTools [23]. The structures of the substrate molecules (R)-1 and (S)-1 were generated in ChemDraw Ultra 9.0 and energy-minimized in Chem3D Pro 9.0. Free rotation of the hydrogen-bonding OH groups in both oxiranepolarizing tyrosines was allowed during substrate docking. The selection of appropriately bound epoxide conformers was based on the presence of two hydrogen bonds with the oxirane-polarizing tyrosines and a proper orientation of the oxirane ring, allowing attack of the nucleophilic aspartate at either carbon atom of the epoxide moiety [24]. 3. Results and discussion 3.1. Phylogenetic analysis The intron-less eph-k2 gene encodes a soluble ␣/␤-hydrolase fold EH of 339 amino acids, which was termed Kau2 [6]. Its closest homologues in the database GenBank are a putative EH (accession number ZP 08629146) from a strain classified as a Bradyrhizobiaceae family member with a protein sequence identity of 85% and a gap of 2 residues, followed by putative EHs from various Rhodopseudomonas palustris and Bradyrhizobium sp. strains with sequence identities in the range of 71–75% and gaps of 4 residues. The next lower level of homologues is comprised of the sequences of putative EHs from Caulobacter spp. with protein sequence identities of 54–55% and gaps of 14–18 residues. Interestingly, the gaps in

these Kau2 homologues are almost exclusively found in the presumed cap loop of the cap domain (see below). One can speculate that insertions and deletions in this mostly unstructured loop of ␣/␤-hydrolase fold EHs is one of nature’s evolutionary strategies to modify EH specificity without disrupting the overall tertiary fold. Insertions and deletions in the N-terminal part of the cap domain were observed in haloalkane dehalogenases, which are also members of the ␣/␤-hydrolase fold enzyme family [25,26]. A phylogenetic analysis of soluble EH-encoding protein sequences confirms the distinct position of the Kau2 sequence within a clade of bacterial EHs which are related to the soluble EHs (sEHs) of higher organisms (Fig. 1). This clade is part of a larger group of bacterial and environmental sequences (designated M-2) which are clustered around the mammalian sEH clade. Bacterial EHs which are related to the soluble EHs of higher organisms have been previously identified in two large phylogenetic analyses of ␣/␤-hydrolase fold EH sequences [27,28]. 3.2. Homology model construction and inhibitor studies Sequence alignments of Kau2 with five protein sequences of ␣/␤-hydrolase fold EHs whose structures have been solved revealed that three of them share a moderate similarity over the entire Kau2 sequence: the potato, human, and murine EHs with sequence identities of 32, 35, and 37%, respectively. One should note in these alignments a low-homology stretch of approximately 45 amino acids that very likely represents the cap loop. We built three homology models of Kau2 (see Fig. S3 of the Supplementary material) using the crystal structures of these three EHs as templates [29–31]. The three generated homology models were found to have similar folds; a comparison of the models revealed r.m.s. deviations of 3.7–5.3 A˚ for 324 C␣ atoms, with the potato EH-derived model being structurally most different. Significant differences between the model tertiary structures are evident in some short loop regions and in a long unstructured segment, the cap loop, which connects helix 4 and 5 of the cap domain (Table S2 of the Supplementary material). This domain consists of 6 helices and bears the two conserved tyrosine residues which hydrogen bond with the epoxide oxygen. To substantiate these computational results, we synthesized two inhibitor compounds, CDU and CIU, which were previously shown to act as potent competitive inhibitors of murine sEH with inhibition constants Ki of 6.3 and 17 nM, respectively [16]. Using Kau2 as the target EH, we determined competitive inhibition mechanisms for CDU and CIU with Ki values of 19 and 50 nM, respectively (see Fig. S4 of the Supplementary material). It appears that CDU and CIU are tight-binding inhibitors of Kau2. The strong binding of these long-chain alkylurea inhibitors implies a similar spatial arrangement of active site residues between Kau2 and the murine sEH [16]. A second inhibition study using the potato EH revealed weak binding of CDU and CIU with inhibition constants exceeding 0.8 ␮M (see Fig. S5 of the Supplementary material). Taking the sequence alignment and inhibition data together, we opted for the murine EH-based homology model as our guide for selecting appropriate randomization sites. Based on this structural model, the Kau2 EH has a long L-shaped tunnel, with both ends accessible to the solvent and the catalytic residues located at the bend of the “L” (Fig. 2), as was found in the X-ray structure of the murine EH [32]. 3.3. Selection of randomization sites Kau2 is an efficient biocatalyst for the hydrolysis of 1 [6]. The wild-type enzyme exhibits a moderate enantioselectivity (E-value of 27) combined with a considerable degree of enantioconvergence, i.e. 84% eep at complete conversion of the racemic substrate. To further improve the enantioconvergence, we applied a directed

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Fig. 1. Phylogenetic relationships among soluble EH-encoding protein sequences of the ␣/␤-hydrolase fold superfamily. Each sequence is represented by its GenBank accession number. The data set includes sequences of mammalian sEHs (clade Ma), plant EH sequences (clade P), and selected EH sequences of microbial or environmental origin (see clade M-1 and sequences in the group M-2). The position of the Kau2-EH sequence is indicated with an arrow. A star represents an EH with a determined X-ray protein structure: human EH (NP 001970), murine EH (NP 031966), potato EH (AAA81892), a bacterial EH from Agrobacterium radiobacter AD1 (CAA73331), and a fungal EH from Aspergillus niger LCP 521 (CAB59812), which is a border EH, i.e. a soluble enzyme belonging to the microsomal class of EHs. The M-1 clade includes bacterial or fungal sequences of EHs with known substrate specificities (represented by filled circles), whereas the sequences grouped in M-2 encode putative EHs (open circles) and EHs of known substrate specificities. The best BLASTP matches to the Kau2-EH sequence were included in the data set. The bootstrap consensus tree is inferred from 100 replicates. The evolutionary distances were computed using the Poisson correction method. The bar represents 0.1 amino acid substitutions per site.

evolution approach using the Kau2 homology model as a guide to identify target residues for mutagenesis within the substrate binding pocket. Due to the rather low sequence homology, the model needs to be interpreted with caution; nevertheless, it may be useful for visualizing the approximate spatial positions of residues within the active site (Fig. 2). The selection of target residues for mutagenesis was also inspired by computational substrate docking. First, we studied the binding modes of (S)-1 and (R)-1 using the crystal structures of the murine and potato EHs. There were no fundamental differences in the docking conformations of

(S)-1 between these two EHs (with binding energies of about −4.3 and −4.6 kcal mol−1 , respectively); in both cases, (S)-1 was exclusively found in the right segment of the binding pocket (see Fig. 3 for murine EH). On the other hand, the binding modes of (R)-1 were different. While only one cluster of (R)-1 molecules with binding energies of about −4.1 kcal mol−1 was found in the left segment of the murine EH active site (Fig. 3), two (R)-1 clusters were found in the potato EH, each cluster occupying a different segment of the binding cavity with binding energies of about −4.1 and −3.6 kcal mol−1 for the right and left segment, respectively (Fig. S6 of the Supplementary material).

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located in the left segment (sites A, E and G), in the right segment (sites C, D, H, F) and also at the bend (sites B, C and F) of the L-shaped cavity for mutagenesis (Fig. 2). 3.4. Screening for improved enantioconvergence

Fig. 2. View into substrate binding pocket of wild-type Kau2 homology model based on X-ray structure of murine EH. The substrate (S)-1 (in yellow) was docked into the active site using AutoDock 4.0. Residues of sites that were randomized together in one round: site A (magenta), [W110, F113]; site B (dark blue), [H155, I158]; site C (green), [V290, L291]; site D (light blue), [F182, F183]; site E (orange), [L147, L148]; site F (beige), [F161, P193]; site G (purple), [T134, P135]; and site H (beige), [Q192, P193]. The catalytic nucleophile Asp-109, the oxirane-polarizing tyrosines Tyr-157 and Tyr-259, and the general base His-316 are depicted in gray stick representation. The right and left segment and also the bend of the L-shaped substrate binding tunnel are indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Docking (S)-1 into the active site of the Kau2 homology model revealed a single cluster of ligand conformers with the phenyl ring positioned in the right segment of the substrate binding pocket (Fig. 2), as was found in the murine and potato EHs. Docking the slow substrate enantiomer (R)-1 also resulted in one cluster, with the phenyl group positioned in the left segment of the binding pocket. A previous study based on molecular dynamics simulations of murine EH with bound trans-methylstyrene oxide revealed one probable (out of two possible) binding conformations of this substrate in the active site of the enzyme. Stabilizing ␲-interactions between the substrate and enzyme were found in both conformations, with the phenyl group of the substrate positioned either in the left or in the right segment of the binding pocket [24]. Taking all these data into account, it seemed reasonable to target residues

Fig. 3. Docking of either enantiomer of 1 into binding pocket of murine EH results in one binding conformer of (R)-1 in left segment and one conformer of (S)-1 in right segment of the binding pocket. The oxygen atoms of the epoxides are positioned a hydrogen-bond distance away from both oxirane-polarizing tyrosines Tyr-381 and Tyr-465. The catalytic His-523 is visualized in stick representation beneath the surface of the right segment of the binding pocket. The catalytic nucleophile Asp-333 is located beneath the epoxide rings of the docked substrates.

Screening for enhanced enantioconvergence requires a sufficiently rapid determination of eep . As shown in this report, certain vicinal diols can be conveniently analyzed for their ee by chiral GC analysis without derivatization. However, we noticed during the GC-based screening that an accurate determination of small differences in high eep -values was difficult to achieve in the last two rounds of directed evolution; we therefore stopped screening after round H. To confirm the screening results, we re-analyzed the best variant of each round of directed evolution for its regioselectivity coefficients using complete conversions of rac-1 (see Fig. S2 of the Supplementary material). As shown in Table 1, the enantioconvergence increased considerably during laboratory evolution, from 84% for the wild-type EH to 93% for the final evolved variant F:13-B11, which contained five amino acid replacements in total (W110L, F113L, F161Y, P193G and V290 W). Out of eight rounds of iterative laboratory evolution, five were non-productive and did not bring about a further enhancement in enantioconvergence. It appears that high enantioconvergence for 1 as the substrate requires specific amino acid replacements. Specific exchanges were found in several enantioconvergent variants of a particular evolutionary round: e.g. F113L at site A in the first round, V290W in the third round, and P193G at site F in the sixth round (Table 1). Further, the last round of directed evolution at site H with variant F:13-B11 as the template confirmed that the amino acid changes determined in this variant were important for high enantioconvergence. Randomizing residues at site H, which partially overlapped with site F, resulted in enantioconvergent variants (with eep of 93%), which contained again the P193G amino acid exchange (in combination with either no exchange at position 192 or Q192E/Q192K). Enantioconvergence in Kau2 is a consequence of forcing the nucleophilic attack to occur at either the C1 carbon atom of bound (S)-1 or C2 of bound (R)-1 (Scheme 1). The partitioning of (S)1 between the reaction pathways ␣S and ␤S remained virtually unchanged during the evolution; in contrast, the ␤R pathway, which is the main reaction pathway for (R)-1, became even more dominant and reached a value of 97% in the F:13-B11 variant; this is the reason for the enhancement in enantioconvergence (Scheme 1; Fig. 4). Thus, the mutations which were acquired during the laboratory evolution toward enantioconvergence essentially only influenced the regioselectivity of attack by Asp-109 for bound (R)-1. Interestingly, these mutations were introduced in three different locations of the active site cavity: (1) in the left segment (site A), (2) in the lower (site C) and upper part of the bend (site F [F161Y]), and (3) in the right segment of the L-shaped cavity (site F [P193G]). Without an X-ray structure of the final variant, a detailed analysis of the source of the enantioconvergence is too speculative. It should be noted that the enantioselectivity of the final F:13-B11 variant is even lower than in the wild-type enzyme, which is of great practical importance, since high E-values would only prolong the reaction time to reach complete conversion of rac-1. Moreover, the EH variants which served as templates for subsequent rounds of directed evolution, including the F:13-B11 variant, were not impaired in their enzymatic activities (Table 1). Specific reaction conditions can favorably influence the kinetic properties of EHs such as enantioselectivity and enantioconvergence [33–35]. Different reaction temperatures and the addition of Tween 80 were tested with the aim to further increase the enantioconvergence of the final evolved EH variant. As shown in Table 2, an increase in enantioconvergence was observed when decreasing the reaction temperature (see Fig. S7 of the Supplementary material).

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Table 1 Directed evolution of Kau2 EH toward higher enantioconvergence with 1 as substrate: selection of important variants.a Randomization site

Template

EH variant

Introduced mutations

eep [%]b

No. of clones screened

% active variants

E-valuec

Spec. activity [%]c

n.a.

n.a. wtEH A:3H8

wt 4-A7 3-H8 5-B4 29-F11 1-C1 12-A1 13-B11

n.a. W110F, F113L W110L, F113L V290W V290W, L291I P193G P193E F161Y, P193G

84 87 89 91 90 91 92 93

n.a.

n.a.

2200

2

2900

2

23 ± 3 n.d. 75 ± 5 25 ± 2

3100

4

100 90 ± 10 103 ± 10 110 ± 15 125 ± 18 95 ± 10 90 ± 13 105 ± 9

A C

C:5B4

F

n.d. 17 ± 2

Directed evolution pathway: site A (→ site B [2900, 1]) → site C (→ site D [2900, 2] → site E [1000, 16]) → site F (→ site G [2700, 1] → site H [1000, 10]). Non-productive randomization sites are indicated in parentheses, together with the number of screened clones and the percentage of active variants. b Enantiomeric excess of 2 after complete conversion of rac-1 (determined at 28 ◦ C). c The specific activities and E-values were determined at 28 ◦ C with rac-1. Specific activity of whole cells containing the wild-type EH: 3.5 ␮mol (mg of protein)−1 min−1 , which corresponds to 100%. n.a.: not applicable; n.d.: not determined; wt: wild type. a

98.0

The EH from C. crescentus enabled the enantioconvergent production of (R)-2 with an ee-value of 95% (at 25 ◦ C), however, with a less favorable E-value of ∼30. It should be noted that bi-enzymatic approaches have been worked out for the production of (R)-2; these are, however, less convenient and technically more demanding than mono-enzymatic reactions with enantioconvergent EHs [14]. While finishing this report, a newly published laboratory evolution study of the potato EH showed a pronounced shift in R:S-product ratio toward (R)-3-phenylpropane-1,2-diol in a variant obtained after two cycles of directed evolution [37].

96.0 94.0

[%]

92.0 90.0 88.0 86.0

3.5. Enantioselective EH variants

84.0

During the screening for improved enantioconvergence, we encountered several EH variants that did not significantly react with (R)-1 as the sole substrate; the chromatograms of these variants showed a large peak of unreacted epoxide and almost no diol formation. However, they exhibited a high activity with (S)1. These variants were re-analyzed for their enantioselectivities with rac-1. It turned out that the majority of these variants, as expected, exhibited higher enantioselectivities than the wild-type EH with (R)-1 being the slow substrate. The enantioselective variants had E-values exceeding 40 and were detected in the first and third round of directed evolution (Table 3). Although useful for detecting potentially enantioselective variants, the screening test does not unequivocally reveal all variants with higher Evalues. For instance, the evolved EH variant A:3-H8 (see Table 1) exhibited substantial activity with (R)-1 as the sole substrate (comparable to the wild-type EH); however, we later determined a quite high E-value of 75 in the presence of rac-1. Thus, significant activity in both separate reactions with (R)-1 or (S)-1 does not necessarily correlate with low enantioselectivity in the presence of a racemic substrate; this indicates a complex interaction of the substrate and product enantiomers with the active site residues during catalysis, which consequently has an effect on the E-value-determining specificity constants kcat /KM for both substrate enantiomers. Further, two potentially enantioselective variants with almost no activity with (R)-1, but high activity with (S)-1 as the sole substrate were detected in round E of the screening; however, their E-values (determined with rac-1) turned out to be low (Table 3). A lack of strict correlation between the rates determined with enantiopure substrates and enantioselectivities determined with racemic substrates has been previously reported with EHs [38]. The observed increase in enantioselectivity in the C:14-H6 variant (see Table 3) prompted us to test these amino acid replacements in the wild-type EH and the A:22-C4 variant, which was already shown to have an improved E-value (Table 3). While replacing Val-290 with tyrosine and Leu-291 with valine had a significant

82.0 wt

A

C

F

Fig. 4. Comparison of eep values (♦; after complete conversion of rac-1 at 28 ◦ C) and regioselectivity coefficients ˛S () and ˇR () between wild-type EH and best variant of each productive round of directed evolution. For a definition of the regioselectivity coefficients, see Scheme 1. The following relationships hold: ˛R + ˇR = 100%, and ˛S + ˇS = 100%. The regioselectivity coefficients were determined in complete biotransformation reactions using rac-1 as the substrate (see Section 2 and Fig. S2 of the Supplementary material).

On the other hand, the addition of Tween 80 (in the range of 0.1–1%, vol.) had no effect on the enantioconvergence. At 8 ◦ C an ee-value of 97% for (R)-2 was determined after the complete conversion of rac-1. The wild-type EH from Caulobacter crescentus strain CB15 is an interesting enantioconvergent biocatalyst accepting styrene oxide and rac-1 [36]. The enzyme exhibits a sequence identity of 55% to Kau2; after sequence alignment, five gaps in the sequence of the EH from C. crescentus have been identified; the two largest gaps were found between the Kau2 residues 192–197 and 202–210. Table 2 Influence of reaction conditions on specific activity, E-value, and regioselectivity coefficients using the final variant F:13-B11.a Reaction conditionb 28 ◦ C, (–) 28 ◦ C, (+) 18 ◦ C, (–) 8 ◦ C, (–)

Specific activity [%] 100 83 75 28

E-value

˛S [%]

ˇR [%]

eep [%]c

17 20 20 30

96.0 96.5 97.6 98.4

97.0 96.4 97.3 98.4

93 93 95 97

a For a definition of the regioselectivity coefficients, see Scheme 1. Whole cells were used with rac-1 as substrate. See Fig. S7 of the Supplementary material. b (+): addition of 0.25% (vol.) Tween 80; (−): without Tween 80. c After complete conversion of rac-1.

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M. Kotik et al. / Journal of Molecular Catalysis B: Enzymatic 91 (2013) 44–51

Table 3 Potentially enantioselective Kau2 variants selected during screening.a Randomization site

Template

A

wtEH

C E a b c

A:3-H8 C:5B4

EH variant

Introduced mutations

E-valueb

Spec. activity [%]c

20-F11 20-G4 22-C4 14-H6 11-E10 11-H12

W110A, F113G W110R, F113E W110A, F113R V290Y, L291V L147A, L148E L147Q, L148E

45 ± 5 50 ± 5 65 ± 20 65 ± 15 7±1 7±1

81 90 92 79 75 71

Acknowledgements

Template

EH variant

Introduced mutations

E-valuea

Spec. activity [%]b

wt-EH A:22-C4c

wt-sd 22-C4sd

V290Y, L291V

130 ± 20 50 ± 10

89 ± 15 74 ± 12

a

c

11 10 15 9 6 7

The variants were detected during the GC-based screening for improved enantioconvergence using two separate activity measurements with either enantiomer of 1. E-values were determined at 28 ◦ C with rac-1. The specific activity of the wild-type enzyme corresponds to 100%.

Table 4 Constructed Kau2 variants using site-directed mutagenesis.

b

± ± ± ± ± ±

E-values were determined at 28 ◦ C with rac-1. The specific activity of the wild-type enzyme corresponds to 100%. See Table 3.

enantioselectivity-enhancing effect by a factor of five in the wildtype EH, the same amino acid changes slightly decreased the enantioselectivity in the A:22-C4 variant (Table 4). Interestingly, the diol product of the highly enantioselective variant wt-sd exhibited a high ee-value of 97% at 50% conversion of the racemic epoxide, offering the possibility to isolate both residual (R)-1 and the product (R)-2 in high ee. The computational docking data imply that there are probably two different binding sites for (R)-1 and (S)-1 in the substrate binding pocket of Kau2. It appears that our experimental data are consistent with this hypothesis, since an enhancement in enantioselectivity was only observed with randomization sites near the bend (sites 290–291) or in the left segment (sites 110 and 113) of the substrate tunnel, where the slow (R)-1 enantiomer is thought to be bound during catalysis. The crude extract activities of the enantioselective EH variants were all slightly lower (by 8–26%) than the wild-type enzyme activities (see Tables 2 and 3). 4. Conclusion Sequence alignment and inhibitor binding data in conjunction with homology modeling suggested that the metagenome-derived Kau2 EH is structurally most related to the eukaryotic sEH from mouse, which has a large two-segment substrate binding cavity. Based on a structure model, we performed a directed evolution study by randomizing selected sites within the substrate binding pocket of Kau2. During the screening we found several highly active EH variants with higher enantioselectivity or enhanced enantioconvergence. The variant wt-sd had a 5-fold improved enantioselectivity compared to the wild-type EH; in addition, it exhibited a very high proportion of ˛S -attack, resulting in the formation of (R)-2 with an ee-value of 97% at half-maximum conversion. The final evolved enantioconvergent variant F:13-B11 (containing five amino acid replacements) transformed in a complete conversion at 28 ◦ C rac-1 into (R)-2 with an ee-value of 93%. A further increase in enantioconvergence was observed when decreasing the reaction temperature. We conclude from this study that protein structure modeling together with directed evolution has proved to be a feasible strategy for improving the metagenomederived Kau2 EH. The generated variants open up convenient routes to the manufacture of the chiral building blocks (R)-1 and (R)2 in an almost enantiopure form from the inexpensive racemic epoxide 1.

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