A Smart Library of Epoxide Hydrolase Variants and the Top Hits for Synthesis of (<i>S</i>)‐β‐Blocker Precursors

Kong, Xiangxing; Ma, Qian; Zhou, Jiahai; Zeng, Bu‐Bing; Xu, Jian‐He

doi:10.1002/anie.201402653

Cited by 50 publications

(27 citation statements)

References 30 publications

(6 reference statements)

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“…Of special relevance is conformational state 3 F128A , not observed in the wild‐type enzyme, that presents a wide active site pocket ready to bind NGE and facilitate the product release. The partial unfolding of Tyr144 α‐helix observed in our MD simulations is in line with the experimental observation that doubly mutated F128S/M145S variant presents severe protein folding problems . To further evaluate our hypothesis, we decided to computationally evaluate the M145S variant.…”

Section: Figuresupporting

confidence: 78%

“…Alanine scanning experiments at two positions located in the substrate entrance and product release zones, for example, residues F128 and M145, were used to support the hypothesis of the different active site tunnels . Interestingly, mutation of both positions to smaller residues (serine and alanine) was found to be crucial for enhancing the catalytic activity of the enzyme towards the resolution of bulky epoxide substrates such as naphthyl glycidyl ether ( NGE , the activity is from 25 to 434 times higher than for the wild‐type enzyme) ,. This increase in activity was attributed to the wider product release zone 3 of the variants that is supposed to facilitate the product dissociation step …”

Section: Figurementioning

confidence: 99%

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Epoxide Hydrolase Conformational Heterogeneity for the Resolution of Bulky Pharmacologically Relevant Epoxide Substrates

Serrano-Hervás

Casadevall

Garcia‐Borràs

et al. 2018

Chemistry A European J

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The conformational landscape of Bacillus megaterium epoxide hydrolase (BmEH) and how it is altered by mutations that confer the enzyme the ability to accept bulky epoxide substrates has been investigated. Extensive molecular dynamics (MD) simulations coupled to active site volume calculations have unveiled relevant features of the enzyme conformational dynamics and function. Our long-timescale MD simulations identify key conformational states not previously observed by means of X-ray crystallography and short MD simulations that present the loop containing one of the catalytic residues, Asp239, in a wide-open conformation, which is likely involved in the binding of the epoxide substrate. Introduction of mutations M145S and F128A dramatically alters the conformational landscape of the enzyme. These singly mutated variants can accept bulky epoxide substrates due to the disorder induced by mutation in the α-helix containing the catalytic Tyr144 and some parts of the lid domain. These changes impact the enzyme active site, which is substantially wider and more complementary to the bulky pharmacologically relevant epoxide substrates.

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Section: Figuresupporting

confidence: 78%

Section: Figurementioning

confidence: 99%

Epoxide Hydrolase Conformational Heterogeneity for the Resolution of Bulky Pharmacologically Relevant Epoxide Substrates

Serrano-Hervás

Casadevall

Garcia‐Borràs

et al. 2018

Chemistry A European J

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“…Moreover, we can carry out gram-scale synthesis of (S)-propranolol with the M145A variant of BmEH. Further studies to generate an EH library by semisaturation mutations on the identified hot spots has broadened the substrate scope to more drug precursors in our laboratory (29).…”

Section: Discussionmentioning

confidence: 99%

Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates

Kong

Yuan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Optically pure epoxides are essential chiral precursors for the production of (S)-propranolol, (S)-alprenolol, and other β-adrenergic receptor blocking drugs. Although the enzymatic production of these bulky epoxides has proven difficult, here we report a method to effectively improve the activity of BmEH, an epoxide hydrolase from Bacillus megaterium ECU1001 toward α-naphthyl glycidyl ether, the precursor of (S)-propranolol, by eliminating the steric hindrance near the potential product-release site. Using X-ray crystallography, mass spectrum, and molecular dynamics calculations, we have identified an active tunnel for substrate access and product release of this enzyme. The crystal structures revealed that there is an independent product-release site in BmEH that was not included in other reported epoxide hydrolase structures. By alanine scanning, two mutants, F128A and M145A, targeted to expand the potential product-release site displayed 42 and 25 times higher activities toward α-naphthyl glycidyl ether than the wild-type enzyme, respectively. These results show great promise for structure-based rational design in improving the catalytic efficiency of industrial enzymes for bulky substrates.epoxide hydrolase | X-ray crystallography | protein engineering | product release | bulky substrate O ptically pure epoxides and the corresponding vicinal diols are valuable chiral building blocks for the production of pharmaceutically active compounds and other fine chemicals (1). Existing approaches for preparing enantiopure epoxides and diols include the asymmetric epoxidation or dihydroxylation of olefin substrates and the resolution of racemic epoxides. These reactions can be accomplished with either chemical catalysts such as chiral salen cobalt complexes and porphyrin manganese adducts or biocatalysts such as monooxygenases and epoxide hydrolases (EHs) (2-4). In the past two decades, EHs have received much attention because they are cofactor-independent enzymes that are "easy to use" for catalyzing the hydrolysis of racemic epoxides to yield highly enantiopure epoxides and vicinal diols (1, 5, 6). However, application of EHs in laboratory and industry was often hindered by their narrow substrate scope, low enantioselectivity, and regioselectivity, or product inhibition (7,8).Many protein-engineering efforts have been made to overcome these drawbacks (9, 10). For example, directed evolution by error-prone PCR or DNA shuffling has been used to enhance the activity and enantioselectivity of EHs (11-13). Structureguided mutagenesis also generated a few EH variants with improved catalytic performance (14-16). The strategy of iterative Combinatorial Active Site-Saturation Test (CAST) combines the rational approach and directed evolution to yield high-quality and small focused mutant libraries for screening EHs with better enantioselectivity (7,17). By mutating residues at the substratebinding site, the substrates of EHs have been expanded to include cyclic meso-epoxides, phenyl glycidyl ether (PGE) derivatives, and other...

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“…The rotation and translation functions were calculated at 2.5 A resolution. All potential space groups were checked for with the solution being found in P6 5 . The rotation solution peak height was 3.8 r and the translation solution peak was 5.6 r. The resulting MR solution was subjected to restrained refinement in REFMAC5 [34] resulting in an R-factor of 40.3% and an R-free of 43.2% after 30 cycles of refinement.…”

Section: Data Collection and Structure Determinationmentioning

confidence: 99%

“…chiral precursors of b-blockers [4,5]. These cofactor-independent enzymes can be applied in enantioselective kinetic resolution processes of racemic epoxides as well as in the stereoselective hydrolysis of meso-epoxides, in many cases with high selectivity and conversion yields [6].…”

Section: Introductionmentioning

confidence: 99%

Discovery and characterization of thermophilic limonene‐1,2‐epoxide hydrolases from hot spring metagenomic libraries

et al. 2015

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The epoxide hydrolases (EHs) represent an attractive option for the synthesis of chiral epoxides and 1,2-diols which are valuable building blocks for the synthesis of several pharmaceutical compounds. A metagenomic approach has been used to identify two new members of the atypical EH limonene-1,2-epoxide hydrolase (LEH) family of enzymes. These two LEHs (Tomsk-LEH and CH55-LEH) show EH activities towards different epoxide substrates, differing in most cases from those previously identified for Rhodococcus erythropolis (Re-LEH) in terms of stereoselectivity. Tomsk-LEH and CH55-LEH, both from thermophilic sources, have higher optimal temperatures and apparent melting temperatures than Re-LEH. The new LEH enzymes have been crystallized and their structures solved to high resolution in the native form and in complex with the inhibitor valpromide for Tomsk-LEH and poly(ethylene glycol) for CH55-LEH. The structural analysis has provided insights into the LEH mechanism, substrate specificity and stereoselectivity of these new LEH enzymes, which has been supported by mutagenesis studies. DatabaseThe atomic coordinates and structure factors of the crystal structures have been deposited in the Protein Data Bank with the codes 5AIF (Tomsk-LEH native structure), 5AIG (Tomsk-LEH valpromide complex), 5AIH (CH55-LEH native structure) and 5AII (CH55-LEH PEG complex). Nucleotide sequence data are available in the GenBank databases under the accession numbers KP765711 (Tomsk-LEH) and KP765710 (CH55-LEH).Abbreviations CH55-LEH, limonene-1,2-epoxide hydrolase from the metagenomic DNA from the Chinese sample; de, diastereomeric excess; ee, enantiomeric excess; EH, epoxide hydrolase; LEH, limonene-1,2-epoxide hydrolase; Mt-LEH, Mycobacterium tuberculosis limonene-1,2-epoxide hydrolase; PEG, poly(ethylene glycol); Re-LEH, Rhodococcus erythropolis limonene-1,2-epoxide hydrolase; Tomsk-LEH, limonene-1,2-epoxide hydrolase from the metagenomic DNA from the Tomsk sample.

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A Smart Library of Epoxide Hydrolase Variants and the Top Hits for Synthesis of (S)‐β‐Blocker Precursors

Cited by 50 publications

References 30 publications

Epoxide Hydrolase Conformational Heterogeneity for the Resolution of Bulky Pharmacologically Relevant Epoxide Substrates

Epoxide Hydrolase Conformational Heterogeneity for the Resolution of Bulky Pharmacologically Relevant Epoxide Substrates

Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates

Discovery and characterization of thermophilic limonene‐1,2‐epoxide hydrolases from hot spring metagenomic libraries

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