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Table of Contents
Abstract
1. Introduction
2. Materials And Methods
2.1. Materials
2.2. Construction Of CP-176 Mutant Libraries By Error-Prone PCR
2.3. Site-Directed Saturation Mutagenesis
2.4. Libraries Screening
2.5. Activity Assay Of The Variants Towards (R,S)-DmCpCe
2.6. Kinetic Parameter Determination
2.7. Enzymatic Resolution Of (R,S)-DmCpCe By CP-176 And Its Variants
2.8. Measurements Of The Half-Life And Tm
2.9. Structural Analysis
3. Results
3.1. Random Mutagenesis Of The Circularly Permutated Esterase CP-176
3.2. Site-Directed Saturation Mutagenesis At The Two Hot Spots
3.4. Importance Of Mutation G282S For Improving CP-M1 Thermostability
3.5. Enzymatic Resolution Of (R,S)-DmCpCe Comparatively With CP-176 And Its Variants
4. Discussion
5. Conclusion
Acknowledgments
Appendix A. Supplementary Data
Abstract
A pharmaceutically relevant esterase, RhEst1, could catalyze the hydrolysis of (R,S)-ethyl-2,2-dimethyl cyclopropane carboxylate [(R,S)-DmCpCe] with excellent enantioselectivity, producing (S)-(+)-2,2-dimethyl cyclopropane carboxylic acid [(S)-DmCpCa], which is a key chiral building block for the synthesis of Cilastatin. In our previous work, a mutant RhEst1-M2 was identified with 6.4-fold higher activity than the wild-type. Additionally, the termini of RhEst1 protein were altered by circular permutation (CP), resulting in a mutant CP-176 which still maintains the catalytic activity of esterase. In this work, to improve the catalytic properties of RhEst1, the mutant CP-176 was taken as the parent of directed evolution. Consequently, a new mutant designated as CP-M1 (=CP-176G282S) was identified, indicating 3.2-fold catalytic efficiency enhancement and nearly 7°C improvement in melting temperature (Tm) as compared with CP-176. Furthermore, the beneficial mutation “G282S” of CP-M1 was reversely introduced into RhEst1-M2, generating the best mutant M3 (=RhEst1-M2G167S), with 1.8fold catalytic efficiency improvement and nearly 10°C improvement of Tm, as compared with RhEst1-M2. This is the first report that the circular permutation and random mutagenesis were combined to reshape a protein, affording distinctly improved activity and thermostability.
Contents lists available at ScienceDirect Molecular Catalysis journal homepage: www.elsevier.com/locate/mcat Protein termini relocation plus random mutation: A new strategy for finding key sites in esterase evolution Yi-Ke Qia, Fu-Long Lia, Qi Chena, Zhi-Jun Zhanga, Zheng-Jiao Luana, Jian-He Xua,b, Hui-Lei Yua,b,⁎ a State Key Laboratory of Bioreactor Engineering, PR China b Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai, 200237, PR China A R T I C L E I N F O Keywords: Biocatalysis Enzymatic resolution Esterase Circular permutation Directed evolution Cilastatin A B S T R A C T A pharmaceutically relevant esterase, RhEst1, could catalyze the hydrolysis of (R,S)-ethyl-2,2-dimethyl cyclopropane carboxylate [(R,S)-DmCpCe] with excellent enantioselectivity, producing (S)-(+)-2,2-dimethyl cyclopropane carboxylic acid [(S)-DmCpCa], which is a key chiral building block for the synthesis of Cilastatin. In our previous work, a mutant RhEst1-M2 was identified with 6.4-fold higher activity than the wild-type. Additionally, the termini of RhEst1 protein were altered by circular permutation (CP), resulting in a mutant CP-176 which still maintains the catalytic activity of esterase. In this work, to improve the catalytic properties of RhEst1, the mutant CP-176 was taken as the parent of directed evolution. Consequently, a new mutant designated as CP-M1 (=CP-176G282S) was identified, indicating 3.2-fold catalytic efficiency enhancement and nearly 7℃ improvement in melting temperature (Tm) as compared with CP-176. Furthermore, the beneficial mutation “G282S” of CP-M1 was reversely introduced into RhEst1-M2, generating the best mutant M3 (=RhEst1-M2G167S), with 1.8- fold catalytic efficiency improvement and nearly 10℃ improvement of Tm, as compared with RhEst1-M2. This is the first report that the circular permutation and random mutagenesis were combined to reshape a protein, affording distinctly improved activity and thermostability.
1. Introduction
Cilastatin, combining with carbapenem antibiotic Imipenem is essential for the clinical treatment of serious infected patients [1–3]. (S)(+)-2,2-Dimethyl cyclopropane carboxylic acid (abbreviated hereafter as (S)-DmCpCa) is a key chiral building block for the synthesis of Cilastatin. Highly efficient and enantioselective synthesis of optically pure organic acids, especially (S)-DmCpCa, represents a greater challenge than the synthesis of chiral alcohols by either chemical or enzymatic methods [4]. Various strategies have been developed recently. Enzymatic preparation of chiral synthons provides a direct, efficient, green, and highly chemoselective/enantioselective alternative, due to the excellent catalytic properties of diverse enzymes. An α/β-fold hydrolase RhEst1 from Rhodococcus sp. strain ECU1013, could catalyze the asymmetric hydrolysis of racemic ethyl (R,S)-2,2-dimethyl cyclopropane carboxylate [(R,S)-DmCpCe] to produce (S)-DmCpCa [4,5] (Scheme 1), and is the first reported esterase with such catalytic function. However, enzymatic characterization of RhEst1 indicated that this enzyme showed relatively low catalytic activity towards the unnatural substrate DmCpCe, which is a principal bottleneck against industrial application. Therefore, various engineering strategies were proposed and adopted for improving the activity of RhEst1. Directed evolution as an effective engineering-strategy was frequently applied to improve the performance of an enzyme, such as activity, thermostability and substrate specificity [6–10]. In our previous work, a quadruple mutant, RhEst1-M2 (RhEst1A143T/A147I/V148F/ G254A), with 6.4-fold higher activity than the wild-type was obtained by substrate channel evolution plus random mutagenesis [11] and cap domain engineering [12]. Nevertheless, there was no significant improvement even if additional random mutagenesis was further performed. The variant RhEst1-M2 seems to have little improvement potential since it might become stuck in a local optimum of the evolution landscapes [13]. Circular permutation (CP) is a typical protein engineering method also inspired from nature [14,15], which rearranges the order of polypeptide sequence by altering the locations of new termini. We also tried to tailor RhEst1 with the inspiring strategy [16], resulting in a functionally active CP variant, CP-176, where the number “176” indicates the cleavage site of circular RhEst1 sequence which is located in the loop region between α5 and α6 helices of RhEst1 protein. Unfortunately, the catalytic activity of CP-176 towards DmCpCe declines to 0.10 U/mg protein from 0.17 U/mg protein of native RhEst1, https://doi.org/10.1016/j.mcat.2018.08.018 Received 12 June 2018; Received in revised form 21 August 2018; Accepted 25 August 2018 ⁎ Corresponding author at: State Key Laboratory of Bioreactor Engineering, PR China. E-mail address: huileiyu@ecust.edu.cn (H.-L. Yu). Molecular Catalysis 460 (2018) 94–99 2468-8231/ © 2018 Elsevier B.V. All rights reserved. T and its thermostability also decreases significantly [16]. However, it was believed that the decline of CP-176 activity might move into troughs before navigating a new higher peak due to the nature of rugged fitness evolution landscapes [13]. Considering both random mutagenesis and circular permutation are effective protein engineering strategies inspired from nature, we speculate that the proper combination of the two useful strategies may be beneficial. Hence the new strategy by combining directed evolution with circular permutation was designed. The variant CP-176 with altered termini was employed as a parental protein of directed evolution to reshape the protein structure for improving its catalytic function. Although the resultant mutants did not reach any higher activity peak than RhEst1-M2, a sensitive point (CP-176G282S) hidden previously was uncovered by this cycle of random mutagenesis, with 3.4-fold activity and nearly 7℃ of Tm improvements compared with CP-176. Subsequently, the integrative effect of this positive mutation on RhEst1M2 was further examined. Interestingly, when the beneficial mutation G282S of CP-M1, which is equivalent to G167S in RhEst1, was introduced into RhEst1-M2, the resultant variant RhEst1-M3 gave the best performance, with 80% catalytic efficiency increase and nearly 10℃ improvement of Tm compared with RhEst1-M2, indicating the effectiveness of the newly proposed strategy of circular permutation plus directed evolution.
2. Materials and methods
2.1. Materials
Tryptone and yeast extract were obtained from Oxoid (Shanghai, China). rTaq polymerase, T4 DNA ligase, and PrimeSTARHS were all purchased from TaKaRa (China). Restriction endonucleases (DpnI, EcoRI, HindIII) were purchased from New England Biolabs (Beijing, China). All oligonucleotide primers were synthesized by Generay (Shanghai, China). DNA Purification Kits were purchased from Tiangen (Beijing, China) for DNA isolation and purification. Plasmid Extraction Mini Kits were purchased from Favorgen (Taiwan, China) for plasmid extraction. Racemic ester (R,S)-DmCpCe was prepared from the acid (R,S)-DmCpCa and ethanol by chemical esterification in our laboratory [12]. The acid (R,S)-DmCpCa was obtained commercially with an analytical grade (Zhejiang Hisoar Pharmaceutical Co., Ltd.).
2.2. Construction of CP-176 mutant libraries by error-prone PCR
To obtain mutated CP-176 gene, error prone PCR was carried out on the CP-176 gene (873 bp) and the amino acid mutagenesis rate was controlled at 0.3%–0.7%. For amplifying the CP-176 gene, the forward primer (containing an EcoRI restriction site) and the reverse primer (containing a HindIII restriction site) are as follows: 5′-ATCCGGAATTCGAAGGGTGCAGCGTCGCAG-3′ 3’-GAGTGGCCGGGTGGGCTCCCGTTCGAACCTT-5′ The PCR system contained 60 ng template plasmids (pRSFDute-CP176), dNTP mix (0.2 mM each), 0.2 μM of each primer, 150 μM Mn2+, PCR buffer and 2.5 U rTaq polymerase in a total volume of 50 μL. The PCR was implemented under the following conditions: initial heating at 95℃ for 3min then 35 cycles of heating at 96 ℃ for 30 s, 57℃ for 40 s and 72℃ for 1min, followed by an extension step at 72℃ for 10min. The PCR products were separated by agarose gel electrophoresis and purified by using a DNA recovery kit (Qiagen, shanghai), digested with EcoRI and HindIII, and ligated into pRSF-Dute plasmid which was also digested with the same two enzymes.The recombined vector was transformed into E. coli BL21 (DE3) competent cells, and plated onto LB agar medium with 50 μgmL−1 of kanamycin.
2.3. Site-directed saturation mutagenesis
For site-directed saturation mutagenesis, the codon of corresponding target amino acid was replaced by NNK degenerate codon. The PCR amplification products were treated with DpnI for 2 h to digest the template plasmids before being transformed into the competent cells. Colonies harboring the CP-176 mutant genes were transferred into 96-deep well plates and cultivated at 37 ℃ and 220 rpm for 12 h, with each well containing 300 μL LB fresh medium and 50mg L−1 kanamycin. These plates were preserved as master plates with glycerol at a final concentration of 10% (v/v).
2.4. Libraries screening
The libraries screening was conducted for finding the variants with improved activity compared with CP-176 by measuring the increase in absorbance of p-nitrophenol at 405 nm with a Microplate Reader [4]. The reaction mixture was consisted of 1mM pNPP (p-nitrophenol propionate) and an appropriate amount of cell free extract in KPB (potassium phosphate buffer, pH 8.0,100mM).
2.5. Activity assay of the variants towards (R,S)-DmCpCe
To determine the enzymatic activity toward (R,S)-DmCpCe, a 500- μL scale reaction containing a certain amount of the purified enzyme (0.1mM or 0.2mM) and 10mM (R,S)-DmCpCe (with 10% v/v of DMSO) was performed at 30℃ and 1000 rpm on a mini-shaker (Eppendorf, Germany) [4]. After a certain period of time, 20 μL of 20% (w/v) H2SO4 was added to terminate the reaction. Then the acidulated product was extracted with an equal volume of ethyl acetate including 0.5 mM dodecane as an internal standard and detected by gas chromatography (GC). The quantitative analysis of the product enantiomers (R,S-DmCpCa) were determined by using a gas chromatography instrument (GC-2014; Shimadzu) equipped with a flame ionization detector and a CP-ChirasilDex CB capillary column (25m by 0.25mm; Varian Co., Palo Alto, CA, USA). The analysis method by GC is the same as described previously [4]. The retention times of (S)-DmCpCa and (R)-DmCpCa were 9.28 and 9.55min, respectively.
2.6. Kinetic parameter determination
The kinetic parameters of the purified variants toward (R,S)DmCpCe were determined by measuring the activity under the varied substrate concentrations (0.05–10mM). More details about the kinetic Scheme 1. RhEst1-catalyzed enantioselective hydrolysis of (R,S)-2,2-DmCpCe. parameter determination were described previously [11].
2.7. Enzymatic resolution of (R,S)-DmCpCe by CP-176 and its variants
Enzymatic resolution [17] of (R,S)-DmCpCe was evaluated in 10-mL scale reactions (30 ℃, 180 rpm), which contained the cell-free lysate of 0.1 g wet cells, 100mM (R,S)-DmCpCe (with 0.5% w/v Tween-80), 100mM KPB, pH 8.0. The reactants (without adding cell-free lysate) were processed by transitory ultrasonic emulsification before bio-resolution, then agitated at 30 °C. At the scheduled times, 50 μl reaction mixture was withdrawn and diluted into 450 μl KPB (100mM, pH 8.0). After 20 μl H2SO4 solution (20%, w/v) was added to terminate the enzymatic transformation, reaction mixture was then extracted by 500 μl ethyl acetate including 0.5 mM dodecane as the internal standard, dried over by anhydrous Na2SO4 and analyzed by chiral GC. Preparative resolution of (R,S)-DmCpCe by the variant was the same as described previously [12], and the result was showed in Table 5.
2.8. Measurements of the half-life and Tm
To test the thermostability of CP-176 and variants, the half-life and Tm of enzymes were determined. The method and details of determination of the half-life and Tm are described previously [12,16].
2.9. Structural analysis
The three-dimensional structure of RhEst1-M2 and RhEst1-M3 were obtained by homology modeling which was carried out by using Modeller with the crystal structure of RhEst1 (PDB ID: 4RNC) as the template. PyMOL (Delano Scientific, Palo Alto, CA, USA) was used to visualize the modeled structure.
3. Results
3.1. Random mutagenesis of the circularly permutated esterase CP-176
As a new starting point of the esterase evolution, CP-176 with altered termini was randomly mutated to improve the catalytic properties. At the beginning of directed evolution, error-prone PCR was applied to generate random libraries for improving the esterase activity towards (R,S)-DmCpCe, based on the heterologous expression system E. coli–pRSFDute-CP-176. After screening of over 4000 variants, two hits, CP-M1 (=CP-176G282S) and CP-M2 (=CP-176A228S), with 3.4-fold and 1.9-fold activity improvements compared with CP-176, were identified respectively (Table 1). Subsequently, the beneficial mutations (G282S and A228S) were simultaneously introduced into CP-176 in order to obtain a better variant. Unfortunately, the double mutant CP-M3 (=CP176A228S/G282S) showed little improvement in specific activity compared with CP-176, and its catalytic activity was even lower than either of the single mutants (Table 1).
3.2. Site-directed saturation mutagenesis at the two hot spots
In spite of the failure in finding any synergistic effect between the two beneficial mutations of A228S and G282S, site-directed saturation mutagenesis was carried out to investigate the individual influence of each mutation site on the catalytic property. Consequently, four more positive mutants in addition to A228S and G282S were further identified, including A228 T, G282W, G282 L and G282 A, with 1.6∼2.1-fold catalytic efficiency (kcat/Km) of wild-type (Table 2). G282 A showed the highest catalytic efficiency improvement (2.1-fold of wild-type) among the G282-mutants, while the mutant A228 T showed the highest increase (1.6-fold of wild type) among the A228-mutants. Interestingly, A228 T is one of the only two positive A228-mutants (including A228S) with higher specific activity than CP-176, indicating that the site of G282 is more sensitive than A228 for activity improvement. As for the four positive G282-mutants, the activity improvements of G282W, G282 L and G282 A were mainly due to the significant increase of kcat, and their Km values were all higher than WT; while the activity improvement of CP-M1 (G282S) was a result of integrated effect of Km and kcat, both Km and kcat of G282S have preferred changes compared with CP-176. Hence, such a potentially beneficial mutation (CP176G282S) that was ignored previously, with 3.4-fold specific activity improvement (3.2-fold catalytic efficiency improvement) was revealed and re-evaluated through directed evolution based on error-prone PCR mutagenesis and site-directed saturation mutagenesis starting from CP176. 3.3. Translation of beneficial mutations from CP-M1 into RhEst1-M2 As aforementioned, CP-176 was resulted from the circular permutation of native RhEst1, where the cleavage site of circular RhEst1 is located on the loop region between α5 and α6 helices of RhEst1 protein. It is because of the highly flexibility of this loop that the variant CP-176 is able to remain functionally active. Hence it is presumed that CP-176 may maintain the necessary integrity of the catalyst’s core region as that of RhEst1. To explore combinatorial role of the positive mutations originated respectively from CP-M1 and RhEst1-M2, we performed the corresponding translation of mutation sites. Firstly, the beneficial mutation of “A143T/A147I/V148F/G254A” which reshapes the substrate channel and increases the soluble expression level of RhEst1 [12], equivalent to a putative mutation of “G78A/A258T/A262I/V263F” in CP-176, was introduced into CP-M1. The resultant variant CP-M4 (= CP-176 G78A/A258T/A262I/V263F/G282S) showed nearly 14-fold higher catalytic efficiency than CP-176 and 4- fold higher catalytic efficiency than CP-M1 (Table 3), reaching the comparable activity level of RhEst1-M2. These results indicate that the major structure of the catalysis-related regions in CP-176 should remain highly similar to that of RhEst1, suggesting that either of the two circularly permuted but structurally similar proteins could probably be reshaped by translating the equivalent mutations from one to the other. Subsequently, the positive mutation G282S in CP-M1, corresponding to a putative mutation G167S in RhEst1, was introduced into RhEst1-M2. The resultant variant M3 (i.e., RhEst1-M2G167S) improved the activity by 50% and elevated Tm by nearly 10 ℃ as compared to those of RhEst1-M2 (Table 3).
3.4. Importance of mutation G282S for improving CP-M1 thermostability
Thermostability is one of the most important properties of an enzyme, especially true for those of industrial importance. Hence we evaluated the kinetic and thermodynamic stabilities of CP-176 and its variants by measuring both the half-life and the melting point Tm. As shown in Table 4, the half-life of CP-176 at 50℃ is only 0.14 h, much shorter than that of RhEst1 (0.37 h), mainly due to the change of structure caused by circular permutation. Surprisingly, both the halflives of CP-M1 and RhEst1-M3 are distinctly higher than those of CP176 and RhEst1-M2 respectively, indicating that the G282S mutation in CP-176 or the G167S mutation in RhEst1-M3 is extremely beneficial for improving their thermostability. To further explore the relationship between thermostability and protein structure, the melting temperature (Tm) of the polypeptide was also measured by circular dichroism (CD) spectroscopy. As a result, introduction of mutation G282S into CP-176 or G167S into RhEst1-M2, elevates the Tm of CP-M1 by 10.6℃ as compared with that of CP-176, and the Tm of RhEst1-M3 by 9.1℃ in comparison to that of RhEst1-M2, indicating the overall structural stability of proteins CP-M1 and RhEst1M3 is significantly improved.
3.5. Enzymatic resolution of (R,S)-DmCpCe comparatively with CP-176 and its variants
The time courses of (R,S)-DmCpCe resolution using CP-176 and its variants were comparatively evaluated by performing enzymatic reactions in 10-mL scale. The progress curves of the reaction course are shown in Fig. 1. After 12 h of reaction, the conversion of CP-176 system reached 24% with an eep of 94%; at the same time, the conversion of CP-M1 system was 46% with an eep of 99%, indicating the distinctly higher catalytic efficiency of CP-M1 than CP-176. As for CP-M4 and CPM5 (= CP-176 G78A/A258T/A262I/V263F), the introduction of G282S mutation also contributes to the improvement of CP-M4 activity over CPM5. It is obvious that RhEst1-M3 was the best variant for biocatalytic resolution of (R,S)-DmCpCe, giving 47% conversion at 2 h with an eep of 98%. As for preparative resolution of (R,S)-DmCpCe by RhEst1-M3, with 500mM substrate and 4 g/L catalyst loading, the conversion reached 46% after only 4 h with an eep of 97% (Table 5), better than any other mutants reported before.
4. Discussion
The crystal structure of RhEst1 (PDB ID: 4RNC) with 1.9 Å resolution was determined by the molecular replacement method in previous work [11]. In order to lucubrate the mechanism of the key mutation site “G167S” (that is “G282S” for CP-176) in RhEst1-M3, homology modeling was carried out. According to the homology modeling of RhEst1M3, the mutation site “G167S” is located on the rim of the substrate channel exit, and the hydroxyl group on side chain of Ser167 is facing towards the surface of protein. Serine can form hydrated clusters with surrounding water molecules by hydrogen bonds, and the bond length of the hydrogen bonds ranges from 1.9 Å to 2.0 Å, belonging to the moderately strong bond. The hydroxyl group of Ser167 generates three more hydrogen bonds with molecule H2O on the protein surface than before (G167), strengthening the hydration shell of the protein surface, which may lead to the great improvement of thermostability (Fig. 2). Analogous case has been reported in the engineering of a more thermally stable lactate dehydrogenase, in which the conserved hydrophobic residue Ile250 had been replaced by the more hydrophilic residue asparagine, and the reduction of the hydrophobic surface results in the mutant tetramer being more thermally stable than the wild-type a The purified enzyme was diluted to 0.42mg protein/mL in 10mM KPB (pH 7.0) for Tm measurement by circular dichroism (CD) spectroscopy. enzyme [18]. From the perspective of kinetic parameters, the improved activity of RhEst1-M3 was a result of synergistic effect of both Km and kcat, the Km reduced while the kcat increased with the introduction of mutation G167S. The same went for CP-M1, its Km and kcat were improved compared with CP-176. Even if both the Km and kcat of CP-M1 and RhEst1-M3 were improved, the substrate’s binding affinity (Km) was already relatively high for esterases [19]. And the increase of the turnover rate kcat of mutants was the main reason for activity improvement. As the site 167 is located on the rim of the substrate channel exit, we still have no adequate theory to further explain it, and previous researches also showed that the residues outside of the active site could also influence the enzyme activity [20]. In the aspect of application, except the bioproduction of optically pure (S)-2,2-DmCpCa, RhEst1 has a quite broad substrate scope. As revealed by fingerprinting analysis in our previous work [4], RhEst1 prefers to short-chain caboxylic esters, especially that with a cyclobutanyl group. So the modified esterase can also be used in the enantioselectively hydrolysis of short-chain caboxylic esters for synthesis of optically pure organic acids.
5. Conclusion
Directed evolution is an effective approach to engineer new enzymes. On the other hand, CP offers an exciting new strategy for protein engineering as a platform technology to generate new templates for directed evolution experiments. Here we combined directed evolution with circular permutation, starting the directed evolution with a CP mutant CP-176. The key mutation site “G282S” was identified from a library consisting more than 4× 103 variants, with significant improvement in the specific activity and thermostability. Interestingly, the mutation site “G282S”, which is “G167S” in RhEst1, was introduced to RhEst1-M2, and the resulting RhEst1-M3 was obtained successfully with 1.8-fold catalytic efficiency improvement (50% specific activity improvement) and nearly 10 ℃ improvement of Tm compared with RhEst1-M2. This is the first report that directed evolution and circular permutation are combined to engineer a protein, which could be extended as a positive reference for other enzyme engineering.
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (Nos. 31500592, 21536004 & 21672063) and the Science and Technology Commission of Shanghai Municipality (No. 15JC1400403).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.mcat.2018.08.018.
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