Directed Evolution of an Enantioselective Epoxide Hydrolase: Uncovering the Source of Enantioselectivity at Each Evolutionary Stage

Reetz, Manfred T.; Bocola, Marco; Wang, Li Wen; Sanchis, Joaquı́n; Cronin, Annette; Arand, Michael; Zou, Jiezhong; Archelas, Alain; Bottalla, Anne Lise; Naworyta, A.; Mowbray, Sherry L.

doi:10.1021/ja809673d

Cited by 141 publications

(200 citation statements)

References 104 publications

(82 reference statements)

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“…[76] Dies entspricht einer idealen kinetischen Racematspaltung, eine Schlussfolgerung, die auch durch die experimentellen k cat /K M -Werte von WT-ANEH und LW202 gestützt ist. Zur Prüfung der Frage, wie die (R)-und (S)-konfigurierten Substrate 19 in der schmalen Bindungstasche positioniert sind bzw.…”

Section: Ism Zur Erhöhung Der Thermostabilität Von Enzymenunclassified

Gerichtete Evolution stereoselektiver Enzyme: Eine ergiebige Katalysator‐Quelle für asymmetrische Reaktionen

Reetz

2010

Angewandte Chemie

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130

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Asymmetrische Katalyse spielt eine Schlüsselrolle in der modernen synthetischen organischen Chemie, wobei künstliche Katalysatoren und Enzyme die zwei wichtigsten Optionen darstellen. Die Verwendung von Enzymen in der organischen Chemie und Biotechnologie hat in der zweiten Hälfte des vergangenen Jahrhunderts stark zugenommen, oft blieb jedoch der Anwendungsbereich aus mehreren Gründen eingeschränkt. Dazu gehören begrenzte Substratakzeptanz, geringe oder fehlende Stereoselektivität, unzureichende Stabilität und manchmal Produktinhibierung. Während der vergangenen 15 Jahre wurde die genetische Technik der gerichteten Evolution so weit entwickelt und verfeinert, dass all diese Probleme angegangen und in der Regel gelöst werden können. Sie basiert auf wiederholende Zyklen von Genmutagenese, Expression und Screening (oder Selektion). Der vorliegende Aufsatz fokussiert auf gerichtete Evolution stereoselektiver Enzyme, ein fundamental neuer Ansatz zur asymmetrischen Katalyse. Der Schwerpunkt liegt auf Methodenentwicklung in dem Bestreben, diese Art der Katalysator‐Optimierung einfacher, schneller und effizienter als in der Vergangenheit zu gestalten. Die neueren Methoden bescheren dem Chemiker und dem Biotechnologen robuste und selektive Katalysatoren für eine Vielzahl von nützlichen Anwendungen.

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Section: Ism Zur Erhöhung Der Thermostabilität Von Enzymenunclassified

Gerichtete Evolution stereoselektiver Enzyme: Eine ergiebige Katalysator‐Quelle für asymmetrische Reaktionen

Reetz

2010

Angewandte Chemie

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“…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 styrene oxide-like analogs (18,19).…”

mentioning

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 first step of nucleophilic attack that is mediated by C164 is crucial for determining the hydrolysis efficiency. A sufficiently small through-space distance (d) value between the nucleophilic sulfur of C164 and the cyano carbon atom of the substrate could correspond to a near-attack conformer, as discussed previously for nitrilase and other enzymecatalyzed reactions (14,23), and this distance should be shorter in the case of the preferred enantiomer.…”

Section: Diffusion Of O-chloromandelonitrile Into the Pocket Of Bcj2315mentioning

confidence: 91%

Protein Engineering of a Nitrilase from Burkholderia cenocepacia J2315 for Efficient and Enantioselective Production of ( R )- o -Chloromandelic Acid

Wang

Gao

Sun

et al. 2015

Appl Environ Microbiol

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The nitrilase-mediated pathway has significant advantages in the production of optically pure aromatic ␣-hydroxy carboxylic acids. However, low enantioselectivity and activity are observed on hydrolyzing o-chloromandelonitrile to produce optically pure (R)-o-chloromandelic acid. In the present study, a protein engineering approach was successfully used to enhance the performance of nitrilase obtained from Burkholderia cenocepacia strain J2315 (BCJ2315) in hydrolyzing o-chloromandelonitrile. O ptically pure aromatic ␣-hydroxy carboxylic acids, such as mandelic acid and its derivatives, are widely used as intermediates and resolving agents for the production of many pharmaceutical and agricultural products (1, 2). Among them, (R)-ochloromandelic acid is one of the most preferred chiral building blocks for industrial synthesis of the antithrombotic agent (S)-clopidogrel, commercialized under the brand name Plavix (3, 4).Considering the great importance of (R)-o-chloromandelic acid in pharmaceuticals and the ever-growing demand for green catalytic processes, tremendous efforts have been made to establish enantioselective routes of production (1). Among them, the nitrilase-mediated pathway is a simple and practical approach for the commercial production of (R)-o-chloromandelic acid and its derivatives because it does not involve a cofactor, uses a less-expensive starting material, and theoretically yields 100% of the desired product.However, few nitrilases have been identified with high enantioselectivity and activity toward o-chloromandelonitrile (2, 3, 5-7). This severely reduces the practical applications of nitrilasemediated production of optically pure (R)-o-chloromandelic acid. A nitrilase with high enantioselectivity and specific activity is crucial to fit the manufacturing process and reduce industrial production costs. These requirements may be met by discovering new enzymes or by applying protein engineering to enhance the performance of the existing nitrilases, and convincing successes have been achieved by both methods (8).In our previous study, we identified and characterized a novel arylacetonitrilase, BCJ2315, from Burkholderia cenocepacia strain J2315 (9). BCJ2315 demonstrated high activity toward o-chloromandelonitrile and tolerated up to 50 mM o-chloromandelonitrile, though the o-chloromandelonitrile was highly toxic to the enzyme, demonstrating great potential for production of (R)-ochloromandelic acid under high o-chloromandelonitrile concentrations (4). Unfortunately, BCJ2315 showed relatively low enantioselectivity toward o-chloromandelonitrile, providing the desired product with only 89.2% enantiomeric excess (ee), which reduces its value for industrial production of (R)-o-chloromandelic acid (4).In the present study, we aspired to improve the fitness of BCJ2315 for the hydrolysis of o-chloromandelonitrile to efficiently produce (R)-o-chloromandelic acid. A protein engineering approach was successfully used to improve the enantioselectivity and activity of BCJ2315 by changing the enzyme structu...

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Directed Evolution of an Enantioselective Epoxide Hydrolase: Uncovering the Source of Enantioselectivity at Each Evolutionary Stage

Cited by 141 publications

References 104 publications

Gerichtete Evolution stereoselektiver Enzyme: Eine ergiebige Katalysator‐Quelle für asymmetrische Reaktionen

Gerichtete Evolution stereoselektiver Enzyme: Eine ergiebige Katalysator‐Quelle für asymmetrische Reaktionen

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

Protein Engineering of a Nitrilase from Burkholderia cenocepacia J2315 for Efficient and Enantioselective Production of ( R )- o -Chloromandelic Acid

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