Stereoselective Hydrolysis of Epoxides by re<i>Vr</i>EH3, a Novel <i>Vigna radiata</i> Epoxide Hydrolase with High Enantioselectivity or High and Complementary Regioselectivity

Hu, Die; Tang, Cun-Duo; Li, Chuang; Kan, Tingting; Shi, Xiao-Ling; Feng, Lai; Wu, Minchen

doi:10.1021/acs.jafc.7b03804

Cited by 27 publications

(14 citation statements)

References 48 publications

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“…As shown in Table 4, rac-3a was efficiently and enantioselectively hydrolyzed until its concentration up to 800 mM, producing (R)-3a with ee s over 99.0% and yield from 38.5 ± 1.2 to 40.2 ± 1.5% (a theoretical yield of 50%). However, when rac-3a concentration was raised to 1000 mM, the ee s of (R)-3a was only 74.8 ± 2.1% until 14.0 h. The reason may be that there was a severe inhibition or denaturation effect on EHs when the substrate reached or exceeded a limit concentration 7,22 . Therefore, the maximum allowable concentration of rac-3a was 800 mM.…”

Section: Specific Activities and Kinetic Parameters Of The Purified Pmentioning

confidence: 98%

“…Herein, the d β was defined as the distance between a nucleophilic side-chain oxygen atom of Asp 101 residue in PvEH3 (or PvEH3 E134K/T137P ) and a C β (the less hindered terminal carbon atom in an oxirane ring) of (S)-or (R)-3a, while the l 1 or l 2 was the hydrogen bond length from a hydroxyl group of proton donor (Tyr 150 or Tyr 232 ) to an oxygen atom in the oxirane ring of epoxide. The binding free energy of PvEH3 (or PvEH3 E134K/T137P ) simulatedly docking with (S)-or (R)-3a, which is closely related to the affinity between them, was calculated using the molecular mechanics Poisson−Boltzmann surface area (MM-PBSA) method 22 .…”

Section: Molecular Docking Simulation Of Eh With (S)-or (R)-3a the Imentioning

confidence: 99%

“…As the hydrolytic reaction of rac-3a Analysis of PvEH3 E134K/T137P with obviously improved preference for (S)-3a. The substrate-binding pocket (SBP) of EHs, harboring a cluster of amino acid residues such as a catalytic triad and two proton donors, was located between the α/β and cap domains which are connected by an NC-loop 22 . It was reported that the spatial position and orientation of substrate-binding residues, influencing the catalytic activity and substrate preference, can be changed by specific residue substitutions in the NC-loop and even more distant regions 45,46 .…”

Section: Specific Activities and Kinetic Parameters Of The Purified Pmentioning

confidence: 99%

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Significant improvement in catalytic activity and enantioselectivity of a Phaseolus vulgaris epoxide hydrolase, PvEH3, towards ortho-cresyl glycidyl ether based on the semi-rational design

Zhang

Liu

et al. 2020

Sci Rep

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The investigation of substrate spectrum towards five racemic (rac-) aryl glycidyl ethers (1a-5a) indicated that E. coli/pveh3, an E. coli BL21(DE3) transformant harboring a PvEH3-encoding gene pveh3, showed the highest EH activity and enantiomeric ratio (E) towards rac-3a. For efficiently catalyzing the kinetic resolution of rac-3a, the activity and E value of PvEH3 were further improved by site-directed mutagenesis of selected residues. Based on the semi-rational design of an NC-loop in PvEH3, four single-site variants of pveh3 were amplified by PCR, and intracellularly expressed in E. coli BL21(DE3), respectively. E. coli/pveh3 E134K and /pveh3 T137P had the enhanced EH activities of 15.3 ± 0.4 and 16.1 ± 0.5 U/g wet cell as well as E values of 21.7 ± 1.0 and 21.2 ± 1.1 towards rac-3a. Subsequently, E. coli/pveh3 E134K/T137P harboring a double-site variant gene was also constructed, having the highest EH activity of 22.4 ± 0.6 U/g wet cell and E value of 24.1 ± 1.2. The specific activity of the purified PvEH3 E134K/T137P (14.5 ± 0.5 U/mg protein) towards rac-3a and its catalytic efficiency (k cat /K m of 5.67 mM −1 s −1 ) for (S)-3a were 1.7-and 3.54-fold those (8.4 ± 0.3 U/mg and 1.60 mM −1 s −1 ) of PvEH3. The gram-scale kinetic resolution of rac-3a using whole wet cells of E. coli/pveh3 E134K/T137P was performed at 20 °C for 7.0 h, producing (R)-3a with 99.4% ee s and 38.5 ± 1.2% yield. Additionally, the mechanism of PvEH3 E134K/T137P with remarkably improved E value was analyzed by molecular docking simulation.Enantiomeric isomers of chiral compounds, such as (R)-and (S)-enantiomers of a racemic drug, usually possess different and even antagonistic biological activities and pharmacological functions 1 . Since the early 1990s, there has been an ever-increasing demand for the optically pure epoxides and their corresponding vicinal diols, which are versatile and highly value-added building blocks applied diffusely in the pharmaceutical, fine chemical and agrochemical industries 2,3 . For examples, (S)-styrene oxide (SO) is a crucial drug intermediate for the synthesis of nematocide, anticancer agent -Levamisole, and anti-HIV agent -(−)-Hyperolactone C 4 , while (R)-and (S)-aryl/alkyl glycidyl ethers, such as (S)-benzyl glycidyl ether (1a) and (R)-ortho-cresyl glycidyl ether (3a), for the synthesis of chiral amino alcohols and β-blockers 5 .Along with a green wave of global industrialization, the biocatalysis by whole cells or enzymes, having high enantio-and/or regio-selectivity and little or no byproducts, is recognized as an alternative or supplement to the chemocatalysis that requires hazardous metals and expensive chiral ligands, exampled by Jacobsen's asymmetric ring-opening hydrolysis and epoxidation 6,7 . Epoxide hydrolases (EHs, EC 3.3.2.-), existing widely in organisms These changes in kinetic parameters suggested that the combinatorial substitution, E134K and T137P, in the NC-loop of PvEH3 had a significantly positive effect on its enantiopreference for (S)-3a.

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Section: Specific Activities and Kinetic Parameters Of The Purified Pmentioning

confidence: 98%

Section: Molecular Docking Simulation Of Eh With (S)-or (R)-3a the Imentioning

confidence: 99%

Section: Specific Activities and Kinetic Parameters Of The Purified Pmentioning

confidence: 99%

See 1 more Smart Citation

Significant improvement in catalytic activity and enantioselectivity of a Phaseolus vulgaris epoxide hydrolase, PvEH3, towards ortho-cresyl glycidyl ether based on the semi-rational design

Zhang

Liu

et al. 2020

Sci Rep

Self Cite

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“…To reduce this uncertainty and assist in the interpretation of experimental data, molecular modelling and simulation methods such as protein-ligand docking, molecular dynamics and quantum mechanics are used to describe protein-ligand systems in atomic detail [72]. These methods have been used extensively to provide insight into the selective properties of a number of EHs [24,31,32,74,75] and are applied in Chapters 6 and 7 to better understand the selectivity of AnEH and its mutants.…”

Section: Molecular Modelling For Understanding Protein Propertiesmentioning

confidence: 99%

“…A wealth of structural and biochemical data has been generated for EHs [1,[16][17][18][19][20][21][22][23], however the origin(s) of their enantio-and regioselective (Figure 1.1) properties remains uncertain [24]. EHs therefore continue to be actively studied in order to better understand, predict and improve their selectivities [24][25][26][27][28][29][30][31][32]. This thesis contributes to this endeavour by developing and exploring different, yet complementary, computational methods for the modelling of the enantioselectivity of an EH from Aspergillus niger (AnEH).…”

Section: General Introductionmentioning

confidence: 99%

Computational modelling of enzymes: predicting and understanding the selectivity of an epoxide hydrolase

Zaugg¹

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Enzymes provide improved catalytic efficiency, renewability and higher selectivity compared to traditional chemical synthesis methods. Stereoselective enzymes in particular are highly desired within industry for the production of fine chemicals and drugs. Epoxide hydrolases (EH) are a family of enzymes whose selective properties facilitate the production of valuable intermediates used for the synthesis of many pharmaceuticals. There is a wealth of structural and biochemical data available for EHs, however the origin of their selectivity remains uncertain. Computational and statistical methods can aid in the design and discovery of selective enzymes and interpretation of the experimental data produced during their characterisation. This thesis focuses on the EH from the fungus Aspergillus niger (AnEH) and its aim is two-fold. The first aim is to explore and develop machine learning methods to predict selectivity enhancing mutations for AnEH. The second aim is to get insight into the mechanistic determinants of AnEH selectivity through molecular modelling methods. Publications during candidatureBook chapters • Zaugg J., Gumulya Y., Gillam E. M. J. and Bodén M. (2014) Computational tools for directed evolution: a comparison of prospective and retrospective strategies. Methods in Molecular Biology 1179:315-333 Contributions by others to the thesis Chapter 2 Zaugg J (candidate) and Bodén M researched and wrote the manuscript. Zaugg J, Bodén M, Gumulya Y and Gillam E edited the manuscript. Chapter 3 Zaugg J (candidate) carried out all computational experiments. Gumulya Y provided experimental data. Bodén M and Malde AK provided guidance in experimental design and analysis of results. Chapter 4 Zaugg J (candidate) carried out all computational experiments. Gumulya Y provided experimental data. Bodén M provided guidance in experimental design and analysis of results. Bodén M wrote much of the software (Bnkit) used for creating and training Bayesian networks. Wang Y developed code to allow specification of Dirichlet priors for Bayesian networks. Essebier A provided code for testing Bayesian networks. Chapter 6 Zaugg J (candidate) designed and carried out all computational experiments. Gumulya Y provided experimental data. Chapter 7 Zaugg J (candidate) carried out all computational experiments and designed the protocol used for docking of ligands. Malde AK and Mark AE provided guidance in the design and implementation of molecular dynamics simulations and free energy calculations and the analysis of results. Gumulya Y provided experimental data. Zaugg J, Gumulya Y, Bodén M, Mark AE and Malde AK wrote and edited the manuscript. IX Statement of parts of the thesis submitted to qualify for the award of another degree None. X Research Involving Human or Animal Subjects No animal or human subjects were involved in this research. XI Abstract I List of Figures XVI List of Tables XIX

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Significant improvement of the enantioselectivity of a halohydrin dehalogenase for asymmetric epoxide ring opening reactions by protein engineering

Xue

Zhang

2020

Appl Microbiol Biotechnol

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Stereoselective Hydrolysis of Epoxides by reVrEH3, a Novel Vigna radiata Epoxide Hydrolase with High Enantioselectivity or High and Complementary Regioselectivity

Cited by 27 publications

References 48 publications

Significant improvement in catalytic activity and enantioselectivity of a Phaseolus vulgaris epoxide hydrolase, PvEH3, towards ortho-cresyl glycidyl ether based on the semi-rational design

Significant improvement in catalytic activity and enantioselectivity of a Phaseolus vulgaris epoxide hydrolase, PvEH3, towards ortho-cresyl glycidyl ether based on the semi-rational design

Computational modelling of enzymes: predicting and understanding the selectivity of an epoxide hydrolase

Significant improvement of the enantioselectivity of a halohydrin dehalogenase for asymmetric epoxide ring opening reactions by protein engineering

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