Complete reversal of enantioselectivity of an enzyme-catalyzed reaction by directed evolution

Zha, Dongxing; Wilensek, Stephanie; Hermes, M.; Jaeger, Karl‐Erich; Reetz, Manfred T.

doi:10.1039/b109913m

Cited by 116 publications

(58 citation statements)

References 11 publications

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“…Nevertheless, our studies show that the long-standing dogma regarding the necessity of amino acid substitutions exclusively at the active site to influence enantioselectivity no longer holds (10,36,39,41). Directed evolution is capable of creating structural effects not easily foreseen by traditional approaches.…”

Section: Learning From Directed Evolutionmentioning

confidence: 98%

“…Reversal of enantioselectivity was also achieved (E ϭ 30 in favor of the opposite enantiomer; ref. 39). Several efficient high-throughput ee assays were developed (17,(27)(28)(29)(30)(31)(32), so that today the analytical problems of almost any project on directed evolution of enantioselective enzymes can be solved.…”

Section: Discussionmentioning

confidence: 99%

“…It was therefore an intriguing goal to try to invert the sense of enantioselectivity in the kinetic resolution of ester 1 (11). By screening for the (R)-product and applying similar strategies based on epPCR, saturation mutagenesis, and DNA-shuffling, a mutant showing an (R)-selectivity (E ϭ 30) was in fact evolved (39). It has 11 amino acid substitutions that are quite different from those of the (S)-selective enzyme variants.…”

Section: Figmentioning

confidence: 99%

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Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications

Reetz

2004

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

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319

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A fundamentally new approach to asymmetric catalysis in organic chemistry is described based on the in vitro evolution of enantioselective enzymes. It comprises the appropriate combination of gene mutagenesis and expression coupled with an efficient high-throughput screening system for evaluating enantioselectivity (enantiomeric excess assay). Several such cycles lead to a ''Darwinistic'' process, which is independent of any knowledge concerning the structure or the mechanism of the enzyme being evolved. The challenge is to choose the optimal mutagenesis methods to navigate efficiently in protein sequence space. As a first example, the combination of error-prone mutagenesis, saturation mutagenesis, and DNA-shuffling led to a dramatic enhancement of enantioselectivity of a lipase acting as a catalyst in the kinetic resolution of a chiral ester. Mutations at positions remote from the catalytically active center were identified, a surprising finding, which was explained on the basis of a novel relay mechanism. The scope and limitations of the method are discussed, including the prospect of directed evolution of stereoselective hybrid catalysts composed of robust protein hosts in which transition metal centers have been implanted.

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Section: Learning From Directed Evolutionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

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Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications

Reetz

2004

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

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319

177

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“…So evolvierten wir (R)-selektive PAL-Mutanten mit EFaktoren von 4-5, [9b] und kurz darauf eine Variante mit E = 30 (R). [33] Mit den oben skizzierten Strategien wandten wir uns anderen Enzymen zu, insbesondere Monooxygenasen für enantioselektive Baeyer-Villiger(BV)-Reaktionen [34] und Sulfoxidationen von prochiralen Thioethern.…”

Section: Erste Beispiele Für Gerichtete Evolution Enantioselektiver Eunclassified

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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“…To the best of our knowledge, up to now this is the first reported totally S-selective halohydrin dehalogenase. Enantioselectivity modification has been considered a major challenge in protein engineering, and normally series of mutations are needed in many cases (9,19,20,26). It is quite amazing that such a pronounced effect on enantioselectivity was even accompanied by a 5-to 6-fold improvement in activity, which was produced by a single mutation.…”

mentioning

confidence: 99%

Key Residues for Controlling Enantioselectivity of Halohydrin Dehalogenase from Arthrobacter sp. Strain AD2, Revealed by Structure-Guided Directed Evolution

Tang

Zhu

Zheng

et al. 2012

Appl Environ Microbiol

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bHalohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) is a valuable tool in the preparation of R enantiomers of epoxides and ␤-substituted alcohols. In contrast, the halohydrin dehalogenase from Arthrobacter sp. AD2 (HheA) shows a low S enantioselectivity toward most aromatic substrates. Here, three amino acids (V136, L141, and N178) located in the two neighboring active-site loops of HheA were proposed to be the key residues for controlling enantioselectivity. They were subjected to saturation mutagenesis aimed at evolving an S-selective enzyme. This led to the selection of two outstanding mutants (the V136Y/L141G and N178A mutants). The double mutant displayed an inverted enantioselectivity (from S enantioselectivity [E S ] ‫؍‬ 1.7 to R enantioselectivity [E R ] ‫؍‬ 13) toward 2-chloro-1-phenylethanol without compromising enzyme activity. Strikingly, the N178A mutant showed a large enantioselectivity improvement (E S > 200) and a 5-to 6-fold-enhanced specific activity toward (S)-2-chloro-1-phenylethanol. Further analysis revealed that those mutations produced some interference for the binding of nonfavored enantiomers which could account for the observed enantioselectivities. Our work demonstrated that those three active-site residues are indeed crucial in modulating the enantioselectivity of HheA and that a semirational design strategy has great potential for rapid creation of novel industrial biocatalysts.

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Complete reversal of enantioselectivity of an enzyme-catalyzed reaction by directed evolution

Cited by 116 publications

References 11 publications

Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications

Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications

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

Key Residues for Controlling Enantioselectivity of Halohydrin Dehalogenase from Arthrobacter sp. Strain AD2, Revealed by Structure-Guided Directed Evolution

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