Directed Evolution of Cyclohexanone Monooxygenases: Enantioselective Biocatalysts for the Oxidation of Prochiral Thioethers

Reetz, Manfred T.; Daligault, Franck; Brunner, Birgit; Hinrichs, Heike; Deege, Alfred

doi:10.1002/anie.200460311

Cited by 124 publications

(56 citation statements)

References 38 publications

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“…As an extension of these studies, the original library of 10 000 mutants was screened for enantioselective oxidation of thioethers. 263 For the oxidation of methyl-p-methylbenzyl thioether (158r), wild-type CHMO afforded the (R)-sulfoxide with an ee of 14%. The screen identified more than 20 hits having >85% ee and five mutants with ee > 95% were sequenced (Table 2).…”

Section: Directed Evolutionmentioning

confidence: 99%

Baeyer−Villiger Monooxygenases: More Than Just Green Chemistry

2011

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Section: Directed Evolutionmentioning

confidence: 99%

Baeyer−Villiger Monooxygenases: More Than Just Green Chemistry

2011

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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).…”

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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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“…In the case of longer chain alkanes, the oxidation starts usually in the terminal methyl group to give the primary alcohol, which is further oxidized to the corresponding aldehyde, and finally converted into fatty acid [210,213]. The alkane-activating enzymes are the mono-oxygenases, which are widely used as biocatalysts in several processes [214][215][216]. Temperature is an important factor in these processes, since it affects the solubility of the alkanes and the stability of the enzymes.…”

Section: Potential Existing Technologies and New Considerations For Vmentioning

confidence: 99%

Utilization of Volatile Organic Compounds as an Alternative for Destructive Abatement

et al. 2015

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Abstract:The treatment of volatile organic compounds (VOC) emissions is a necessity of today. The catalytic treatment has already proven to be environmentally and economically sound technology for the total oxidation of the VOCs. However, in certain cases, it may also become economical to utilize these emissions in some profitable way. Currently, the most common way to utilize the VOC emissions is their use in energy production. However, interesting possibilities are arising from the usage of VOCs in hydrogen and syngas production. Production of chemicals from VOC emissions is still mainly at the research stage. However, few commercial examples exist. This review will summarize the commercially existing VOC utilization possibilities, present the utilization applications that are in the research stage and introduce some novel ideas related to the catalytic utilization possibilities of the VOC emissions. In general, there exist a vast number of possibilities for VOC OPEN ACCESSCatalysts 2015, 5 1093 utilization via different catalytic processes, which creates also a good research potential for the future.

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Directed Evolution of Cyclohexanone Monooxygenases: Enantioselective Biocatalysts for the Oxidation of Prochiral Thioethers

Abstract: Mutational changes in cyclohexanone monooxygenases (CHMOs), for example the introduction of serine at position 432, lead to surprisingly versatile enantioselective biocatalysts that perform particularly well in the air‐mediated partial oxidation of prochiral thioethers (see scheme).

Cited by 124 publications

References 38 publications

Baeyer−Villiger Monooxygenases: More Than Just Green Chemistry

Baeyer−Villiger Monooxygenases: More Than Just Green Chemistry

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

Utilization of Volatile Organic Compounds as an Alternative for Destructive Abatement

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