Directed Evolution as a Method To Create Enantioselective Cyclohexanone Monooxygenases for Catalysis in Baeyer–Villiger Reactions

Reetz, Manfred T.; Brunner, Birgit; Schneider, T.; Schulz, Frank; Clouthier, Christopher M.; Kayser, Margaret M.

doi:10.1002/ange.200460272

Cited by 81 publications

(66 citation statements)

References 71 publications

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“…When the alignment of protein sequences of BVMOs included in this study is compared with the recently described point mutations in CHMO Acineto , [27] two striking similarities can be identified. The Leu 143 Phe mutation exactly follows the separation in the two main enzyme groups.…”

Section: Methodsmentioning

confidence: 91%

“…As the authors suggest that the protein undergoes extensive conformational changes in the biocatalytic cycle, further conclusions for distant members of the BVMO family, such as those included in this study, seem rather speculative. However, when we consider the information together with recent results in modifying the enantioselectivity of CH-MO Acineto by random mutagenesis, [27] we can begin to identify BVMO regions with major impact on biocatalytic behavior and stereopreference. Further structural and biotransformation studies on BVMOs more closely related to the two clusters outlined herein and on a larger set of ketonescurrently being addressed in our laboratory-seem necessary for the development of a comprehensive and predictive model for this enzyme family to successfully expand the biocatalytic armament in the field of Baeyer-Villiger oxidations.…”

Section: Methodsmentioning

confidence: 99%

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Family Clustering of Baeyer–Villiger Monooxygenases Based on Protein Sequence and Stereopreference

et al. 2005

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Die Identifizierung von Enzympaaren mit überlappender Substratspezifität und enantiokomplementären Transformationen ist eine zentrale Herausforderung der Biokatalyse. Enantio‐ und regiodivergente Baeyer‐Villiger‐Oxidationen gelangen mit einer kleinen Bibliothek rekombinanter Escherichia‐coli‐Stämme, die Monooxygenasen unterschiedlichen mikrobiellen Ursprungs exprimieren (siehe Bild). Die auf der Stereopräferenz basierende Gruppenbildung von Enzymen stimmt gut mit der phylogenetischen Nähe überein.

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Section: Methodsmentioning

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Family Clustering of Baeyer–Villiger Monooxygenases Based on Protein Sequence and Stereopreference

et al. 2005

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“…These studies tend to reach remarkably similar conclusions about the fractions of mutations that fall into each of these 3 classifications, despite applying different methodologies to different proteins to optimize different properties. Typically, Ϸ30-50% of random mutations are strongly deleterious (17-19), 50-70% are approximately neutral (17)(18)(19), and perhaps 0.5-0.01% are beneficial (20)(21)(22)(23)(24)(25)(26). These experiments therefore make clear that, in a laboratory context, it is almost always possible to find a substantial number of neutral mutations and usually at least a few that enhance stability or an existing function.…”

Section: Empirical Lessons From the Directed Evolution Of Proteinsmentioning

confidence: 97%

In the light of directed evolution: Pathways of adaptive protein evolution

Bloom¹,

Arnold

2009

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

387

345

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Directed evolution is a widely-used engineering strategy for improving the stabilities or biochemical functions of proteins by repeated rounds of mutation and selection. These experiments offer empirical lessons about how proteins evolve in the face of clearly-defined laboratory selection pressures. Directed evolution has revealed that single amino acid mutations can enhance properties such as catalytic activity or stability and that adaptation can often occur through pathways consisting of sequential beneficial mutations. When there are no single mutations that improve a particular protein property experiments always find a wealth of mutations that are neutral with respect to the laboratory-defined measure of fitness. These neutral mutations can open new adaptive pathways by at least 2 different mechanisms. Functionallyneutral mutations can enhance a protein's stability, thereby increasing its tolerance for subsequent functionally beneficial but destabilizing mutations. They can also lead to changes in ''promiscuous'' functions that are not currently under selective pressure, but can subsequently become the starting points for the adaptive evolution of new functions. These lessons about the coupling between adaptive and neutral protein evolution in the laboratory offer insight into the evolution of proteins in nature.evolutionary engineering ͉ neutral evolution ͉ promiscuous activity ͉ protein stability ͉ enzyme engineering

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“…Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIB 9871 (3) was subjected to random mutagenesis, and mutants with improved enantioselectivity were identified (20,21). In a similar fashion, a BVMO from Pseudomonas fluorescens DSM 50106 was evolved toward increased enantioselectivity (16).…”

mentioning

confidence: 99%

Mapping the Substrate Binding Site of Phenylacetone Monooxygenase from Thermobifida fusca by Mutational Analysis

Dudek

Gonzalo

Pazmino

et al. 2011

Appl Environ Microbiol

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Baeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) from Thermobifida fusca is the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio-or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope.Enzymes have been gaining increasing attention as efficient and selective catalysts to be used in synthetic chemistry. Baeyer-Villiger monooxygenases (BVMOs) comprise a group of enzymes that are particularly interesting for synthetic applications. These biocatalysts employ molecular oxygen as a mild oxidant to oxidize carbonylic compounds. Apart from catalyzing Baeyer-Villiger reactions, BVMOs are capable of oxidizing a range of heteroatoms (e.g., sulfur, nitrogen, and boron). Furthermore, they often perform these reactions with high chemo-, regio-, and enantioselectivity (5, 15, 30). The use of oxygen, which is a cheap and clean oxidant, and their diversity of catalyzed reactions make BVMOs attractive candidates for biocatalytic processes.The identification of phenylacetone monooxygenase (PAMO) in the moderately thermophilic bacterium Thermobifida fusca has brought about a breakthrough in the research on BVMOs (11). PAMO is a thermostable enzyme that can easily be expressed in Escherichia coli and purified. Besides, its crystal structure has been solved as the first structure of a BVMO (17). While PAMO shows excellent stability, even in the presence of organic solvents (6, 28), its substrate specificity is rather restricted. The enzyme accepts mainly small aromatic ketones and sulfides (7, 26), whereas the oxidations of bulkier ketones occur with lower activity and selectivity (27). However, the unique...

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Directed Evolution as a Method To Create Enantioselective Cyclohexanone Monooxygenases for Catalysis in Baeyer–Villiger Reactions

Abstract: Die Richtung und der Grad der Enantioselektivität von Baeyer‐Villiger‐Reaktionen, die durch eine Cyclohexanon‐Monooxygenase katalysiert und durch Luft vermittelt werden (z. B. die Desymmetrisierung von 1) lassen sich durch gerichtete Evolution steuern.

Cited by 81 publications

References 71 publications

Family Clustering of Baeyer–Villiger Monooxygenases Based on Protein Sequence and Stereopreference

Family Clustering of Baeyer–Villiger Monooxygenases Based on Protein Sequence and Stereopreference

In the light of directed evolution: Pathways of adaptive protein evolution

Mapping the Substrate Binding Site of Phenylacetone Monooxygenase from Thermobifida fusca by Mutational Analysis

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