Structure-guided improvement in the enantioselectivity of an Aspergillus usamii epoxide hydrolase for the gram-scale kinetic resolution of ortho-trifluoromethyl styrene oxide

https://doi.org/10.1016/j.enzmictec.2021.109778Get rights and content

Highlights

  • Reshaping AuEH2’s SBP for improving its enantioselectivity towards rac-o-TFMSO.

  • Obtaining a mutant AuEH2R322V/L344C with the largest E of 83 among all the mutants.

  • Kinetic resolution of 200 mM rac-o-TFMSO was conducted by E. coli/Aueh2R322V/L344C.

Abstract

Microtuning the substrate-binding pocket (SBP) of EHs has emerged as an effective approach to manipulate their enantio- or regio-selectivities and activities towards target substrates. Here, the enantioselectivity (enantiomeric ratio, E) of AuEH2 towards a racemic (rac-) ortho-trifluoromethyl styrene oxide (o-TFMSO) was improved via microtuning its SBP. Based on the analysis on the crystal structure of AuEH2, its specific residues I192, Y216, R322 and L344 lining the SBP in close to the catalytic triad were identified for site-saturation mutagenesis. After screening, five single-site mutants were selected with E values elevated from 8 to 12―25 towards rac-o-TFMSO. To further improve E, four double-site mutants were constructed by combinatorial mutagenesis of AuEH2R322V separately with AuEH2I192V, AuEH2Y216F, AuEH2L344A and AuEH2L344C. Among all the mutants, AuEH2R322V/L344C possessed the largest E of 83 with activity of 67 U/g wet cell. The kinetic resolution of 200 mM rac-o-TFMSO was conducted at 0 °C for 5.5 h using 80 mg/mL wet cells of E. coli/Aueh2R322V/L344C, a transformant expressing AuEH2R322V/L344C, retaining (S)-o-TFMSO with 98.4 % ees and 49.3 % yields. Furthermore, the molecular docking simulation analysis indicated that AuEH2R322V/L344C more enantiopreferentially attacks the terminal carbon (Cβ) in the oxirane ring of (R)-o-TFMSO than AuEH2.

Introduction

It has been recognized that the enantiomeric isomers of chiral compounds, such as (R)- and (S)-enantiomers of a chiral drug, commonly have different or even antagonistic bioactivities or pharmacological functions [1]. In view of this, the enantiopure epoxides and the corresponding vicinal (1,2-) diols, which are multi-functional and highly valuable building blocks, have been diffusely applied in pharmaceutical, agrochemical, and fine chemical industries [2]. For examples, a chiral ortho-trifluoromethyl styrene oxide (o-TFMSO) was used for the synthesis of neurotherapeutic azole compounds to treat anxiety, depression, obesity, sleep disorder and neuropathic pain, while (S)-o-chlorostyrene oxide for the synthesis of an antiviral agent EMI39.3 [3,4]. To date, several chemical approaches have been developed to produce chiral intermediates, but suffered from several drawbacks, such as hazardous metal catalysts, costly chiral ligands, and harsh reaction conditions [5]. With the increasing of public awareness of environment protection, the biocatalysis using whole-cells, cell-free extracts or isolated enzymes has been regarded as an alternative or supplement to chemocatalysis [6].

Epoxide hydrolases (EHs), coenzyme-independent biocatalysts, catalyze the enantio- and/or regio-selective hydrolysis of rac-epoxides into optically pure epoxides and/or 1,2-diols, displaying a great application potential in chiral chemical industry [7]. The majority of EHs belong to an α/β-hydrolase fold superfamily, and are mainly classified into microsomal EHs (mEHs, EC 3.3.2.9) and soluble EHs (sEHs, EC 3.3.2.10). They are divided into two main structural regions: the α/β and cap domains, between which an SBP is located. It contains one catalytic triad (Asp―His―Asp/Glu) and two conserved Tyr residues activating the nucleophilic attacks on the Cβ and Cα in the oxirane ring of epoxides via forming hydrogen bonds [8]. Compared with sEHs, mEHs also contain an extra meander region at N-termini, playing a crucial role in dimer formation [9]. Based on the reaction mechanisms of specific EH―epoxide pairs, the hydrolytic reactions of rac-epoxides were carried out in two major pathways: the kinetic resolution retaining single epoxides with high enantiomeric excess (ees) and a limitation of 50 % yields, and the enantioconvergent hydrolysis producing enantiopure 1,2-diols with high eep and up to 100 % theoretical yieldp. It is the most crucial element that EHs used in the former must have large E values towards rac-epoxides, or in the latter high regiocomplementarities for (R)- and (S)-epoxides along with small E values [2].

Over the past few decades, numerous EHs have been applied to the kinetic resolution (i.e., enantioselective hydrolysis) of rac-epoxides. However, only a few known wild-type EHs had large E values and high activities towards ortho-substituted aromatic epoxides, such as ortho-nitrostyrene oxide, presumably as the result of large steric hindrance of ortho-substituents [10,11]. For example, the kinetic resolution of rac-o-TFMSO was carried out by A. niger EH, retaining (S)-o-TFMSO (98 % ees) with only 34 % yields (much lower than theoretical yield), mainly attributing to the small E value of 34 with a preference for (R)-o-TFMSO [12]. Therefore, it is necessary to excavate novel EHs with large E values and high activities, or improve the catalytic performances of existing EHs towards ortho-substituted aromatic epoxides. With the development of gene engineering, the site-saturation mutagenesis along with combinatorial mutagenesis on the key residues lining the SBP of EHs, i.e., the reshaping of SBP, has been applied to improve their E values and/or activities [13,14].

In our previous studies, the kinetic resolution of rac-styrene oxide was conducted using AuEH2 in phosphate buffer (pH 7.0) or n-hexanol/buffer (pH 7.0) [15,16]. Additionally, the crystal structure of AuEH2 (PDB: 6IX4) was solved by X-ray diffraction. However, the pre-experiment result of kinetic resolution of rac-o-TFMSO using E. coli/Aueh2, an E. coli transformant expressing AuEH2, was unsatisfactory owing to its poor enantioselectivity (E value of 8). In this work, to enhance the E value of AuEH2 towards rac-o-TFMSO, its SBP was selected as the research object. Four residues, I192, Y216, R322 and L344, lining the hydrophobic SBP of AuEH2 in close to the catalytic triad (D191―H369―E343) were identified based on the analysis on its crystal structure. A total of 76 single-site mutants were constructed via site-saturation mutagenesis on the four residues. After screening, five mutants with improved E values were selected, and subjected to combinatorial mutagenesis. Among all the tested mutants, AuEH2R322V/L344C possessed the largest E value. The gram-scale kinetic resolution of rac-o-TFMSO at high concentration was carried out using whole-cell of E. coli/Aueh2R322V/L344C in the ice-water bath, producing (S)-o-TFMSO and (R)-ortho-trifluoromethyl phenylethane-1,2-diol (o-TFMPED) with high ee values and yields (Fig. 1). Furthermore, the molecular mechanism of remarkably improved enantiopreference of AuEH2R322V/L344C for (R)-o-TFMSO was analyzed via molecular docking (MD) simulation.

Section snippets

Plasmids, strains, and chemicals

A recombinant plasmid pET-28a-Aueh2 and an AuEH2-expressing E. coli transformant (E. coli/Aueh2) were constructed, and preserved in our lab [15]. PrimeSTAR HS DNA polymerase and Dpn I endonuclease (TaKaRa, Dalian, China) were used for site-saturation mutagenesis and combinatorial mutagenesis on the specific residues lining the SBP of AuEH2, i.e., the microtuning of SBP. pET-28a(+) and E. coli BL21(DE3) (Novagen, Madison, WI) were applied to the construction of recombinant plasmids and the

Identification of the specific residues lining the SBP of AuEH2

The crystal structure of a dimeric AuEH2 with two same subunits was solved at 1.51 Å resolution by X-ray diffraction in our lab. AuEH2 belongs to the mEH subgroup of an α/β hydrolase fold superfamily. Each subunit of AuEH2 comprises 395 amino acid residues, and was structurally divided into three major functional regions: a meander region at the N-terminus (a peptide segment from M1 to S82), an α/β domain (a β-sheet surrounded by a cluster of α-helices) consisting of both the N- and C-terminal

Conclusion

To improve the E value of AuEH2 towards rac-o-TFMSO, its SBP was set as the research object, and then subjected to reshaping via laboratory-directed evolution. Based on the analysis on the crystal structure of AuEH2, four residues lining its SBP in close to the catalytic triad were identified for site-saturation mutagenesis. After screening of a single-site mutation library, five mutants, AuEH2I192V, AuEH2Y216F, AuEH2R322V, AuEH2L344A and AuEH2L344C, were selected with enhanced E values,

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (No. 21676117) and Natural Science Foundation for Youth of Jiangsu Province of China (No. BK20180622). We are grateful to Prof. Xianzhang Wu (School of Biotechnology, Jiangnan University) for providing technical assistance.

References (32)

  • S. Karboune et al.

    Properties of epoxide hydrolase from Aspergillus niger for the hydrolytic kinetic resolution of epoxides in pure organic media

    Enzym. Microb. Technol.

    (2006)
  • P. Saini et al.

    An overview on the enhancement of enantioselectivity and stability of microbial epoxide hydrolases

    Mol. Biotechnol.

    (2017)
  • W.J. Choi

    Biotechnological production of enantiopure epoxides by enzymatic kinetic resolution

    Appl. Microbiol. Biotechnol.

    (2009)
  • A. Li et al.

    Asymmetric epoxidation of alkenes and benzylic hydroxylation with P450tol monooxygenase from Rhodococcus coprophilus TC-2

    Chem. Commun.

    (2014)
  • S. Wu et al.

    Enantioselective hydrolysis of racemic and meso-epoxides with recombinant Escherichia coli expressing epoxide hydrolase from Sphingomonas sp. HXN-200: preparation of epoxides and vicinal diols in high ee and high concentration

    ACS Catal.

    (2013)
  • K.H. Hopmann et al.

    Insights into the reaction mechanism of soluble epoxide hydrolase from theoretical active site mutants

    J. Phys. Chem. B

    (2006)
  • Cited by (0)

    View full text