Designing New Baeyer−Villiger Monooxygenases Using Restricted CASTing

Clouthier, Christopher M.; Kayser, Margaret M.; Reetz, Manfred T.

doi:10.1021/jo0613636

Cited by 104 publications

(62 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The F156 and G157 residues of CPMO have recently been confirmed as hotspots in tuning enantioselectivity of CPMO. [11] In this paper we report the change of PAMO substrate specificity by replacing M446: the M446G PAMO mutant. In addition to an altered substrate specificity, the active-site redesign results in improved enantioselective behavior.…”

Section: Introductionmentioning

confidence: 95%

Altering the Substrate Specificity and Enantioselectivity of Phenylacetone Monooxygenase by Structure‐Inspired Enzyme Redesign

et al. 2007

View full text Add to dashboard Cite

Of all presently available Baeyer-Villiger monooxygenases, phenylacetone monooxygenase (PAMO) is the only representative for which a structure has been determined. While it is an attractive biocatalyst because of its thermostability, it is only active with a limited number of substrates. By means of a comparison of the PAMO structure and a modeled structure of the sequence-related cyclopentanone monooxygenase, several active-site residues were selected for a mutagenesis study in order to alter the substrate specificity. The M446G PAMO mutant was found to be active with a number of aromatic ketones, amines and sulfides for which wildtype PAMO shows no activity. An interesting finding was that the mutant is able to convert indole into indigo blue: a reaction that has never been reported before for a Baeyer-Villiger monooxygenase. In addition to an altered substrate specificity, the enantioselectivity towards several sulfides was dramatically improved. This newly designed Baeyer-Villiger monooxygenase extends the scope of oxidation reactions feasible with these atypical monooxygenases.

show abstract

Section: Introductionmentioning

confidence: 95%

Altering the Substrate Specificity and Enantioselectivity of Phenylacetone Monooxygenase by Structure‐Inspired Enzyme Redesign

et al. 2007

View full text Add to dashboard Cite

show abstract

“…A homology model of cyclopentanone monooxygenase (CPMO) from Comamonas sp. strain NCIMB 9872 (13) based on the structure of PAMO supported a redesign study of CPMO by which the enantioselectivity of the enzyme could be improved (4). Recently, the Reetz group described a few cases in which residues in the active site of PAMO were targeted by semirandom mutagenesis (22,23,33).…”

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

View full text Add to dashboard Cite

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...

show abstract

“…The first step in this direction was undertaken by a structureguided approach based on the use of CASTing in combination with a reduced amino acid alphabet, the BVMO in this case being cyclopentanone monooxgenase (CPMO). 67 A homology model was first generated using the X-ray data of phenyl acetone monooxygenase (PAMO), a thermostable BVMO. 68 On this basis, four CAST residues F156, G157, G449 and F450 in direct neighborhood to the modeled Criegee intermediate in CPMO were identified as putative hot spots.…”

Section: Engineering Site-selectivity Of Baeyer-villiger Monooxygenasesmentioning

confidence: 99%

“…Table 2 Oxidative desymmetrization of prochiral ketones using the CHMO mutant F432S in whole-cell reactions 66 Ser, and Gly). 67 This was the first case of using a reduced amino acid alphabet in directed evolution of stereoselective enzymes. At the time only mini-libraries were screened amounting to only 150 transformants, three substrates being considered, namely 4-methyl-, 4-acetoxy-and 4-tert-butylcyclohexanone.…”

Section: Engineering Site-selectivity Of Baeyer-villiger Monooxygenasesmentioning

confidence: 99%

Enzymatic site-selectivity enabled by structure-guided directed evolution

Wang

Reetz

2017

Chem. Commun.

Self Cite

View full text Add to dashboard Cite

Biocatalytic site-selective (regioselective) organic transformations have been practiced for decades, but the traditional limitations of enzymes regarding narrow substrate acceptance and the often observed insufficient degree of selectivity have persisted until recently. With the advent of directed evolution, it is possible to engineer site-selectivity to suit the needs of organic chemists. This review features recent progress in this exciting research area, selected examples involving P450 monooxygenases, halogenases and Baeyer-Villiger monooxygenases being featured for illustrative purposes. The complementary nature of enzymes and man-made catalysts is emphasized.

show abstract

Designing New Baeyer−Villiger Monooxygenases Using Restricted CASTing

Cited by 104 publications

References 52 publications

Altering the Substrate Specificity and Enantioselectivity of Phenylacetone Monooxygenase by Structure‐Inspired Enzyme Redesign

Altering the Substrate Specificity and Enantioselectivity of Phenylacetone Monooxygenase by Structure‐Inspired Enzyme Redesign

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

Enzymatic site-selectivity enabled by structure-guided directed evolution

Contact Info

Product

Resources

About