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

Mihovilovič, Marko D.; Rudroff, Florian; Grötzl, Birgit; Kapitán, Peter; Šnajdrová, Radka; Rydz, Joanna; Mach, Robert L.

doi:10.1002/ange.200462964

Cited by 33 publications

(15 citation statements)

References 41 publications

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“…PAMO is able to produce both regioisomers, similar to CHMO and unlike CPMO, which generates mainly the "normal" lactone (product 2a) (1,18). The "normal" lactone is produced as the (1S,5R)-enantiomer by PAMO, similar to other BVMOs.…”

Section: Discussionmentioning

confidence: 90%

“…The "normal" lactone is produced as the (1S,5R)-enantiomer by PAMO, similar to other BVMOs. PAMO also generates the (1S,5R)-enantiomer of compound 2b, in contrast to CPMO and CHMO, which provide the (1R,5S)-enantiomer of compound 2b (1,18). The Q152F, A435Y, and A442G mutants have manifested inverted enantioselectivity of product 2b, which makes them similar to CPMO and CHMO with respect to the enantioselective conversion of this substrate.…”

Section: Discussionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 90%

Section: Discussionmentioning

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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“…Based on a smaller BVMO sequence data set, Mihovilovic and coworkers (38) previously proposed two groups of CHMO and CPMO "family clustering" based on their stereopreferences. The CHMO type generally displayed broad substrate acceptance, whereas the CPMO type was more restrictive.…”

Section: Discussionmentioning

confidence: 99%

Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ ³ -4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453

Leisch

Shi

Große

et al. 2012

Appl Environ Microbiol

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ABSTRACTA dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor byPseudomonas putidaATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) inEscherichia coliand determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP+at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP+. A comparison of several crystal forms of OTEMO bound to FAD and NADP+revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (kcat/Km) favors 2-n-hexyl cyclopentanone (4.3 × 105M−1s−1) as a substrate, although its affinity (Km= 32 μM) was lower than that of the CoA-activated substrate (Km= 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.

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“…Model Baeyer-Villiger biooxidation resulting in the production of lactone regioisomers (1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and (1R,5S)-3-oxabicyclo-[3.3.0]-oct-6-en-2-one [27] was initiated by the addition of substrate rac-bicyclo[3.2.0]hept-2-en-6-one in ethanol (volume ratio 1:9) to the reaction medium to a final concentration of 0.5 g/L. The reaction conditions were 25 • C and 150 rpm in an orbital shaker.…”

Section: Repeated Biotransformationsmentioning

confidence: 99%

Polyelectrolyte Complex Beads by Novel Two-Step Process for Improved Performance of Viable Whole-Cell Baeyer-Villiger Monoxygenase by Immobilization

et al. 2017

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A novel immobilization matrix for the entrapment of viable whole-cell Baeyer-Villiger monooxygenase was developed. Viable recombinant Escherichia coli cells overexpressing cyclohexanone monooxygenase were entrapped in polyelectrolyte complex beads prepared by a two-step reaction of oppositely-charged polymers including highly defined cellulose sulphate. Immobilized cells exhibited higher operational stability than free cells during 10 repeated cycles of Baeyer-Villiger biooxidations of rac-bicyclo[3.2.0]hept-2-en-6-one to the corresponding lactones (1R,5S)-3-oxabicyclo-[3.3.0]oct-6-en-3-one and (1S,5R)-2-oxabicyclo-[3.3.0]oct-6-en-3-one. The morphology of polyelectrolyte complex beads was characterised by environmental scanning electron microscopy; the spatial distribution of polymers in the beads and cell viability were examined using confocal laser scanning microscopy, and the texture was characterised by the mechanical resistance measurements.

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

Cited by 33 publications

References 41 publications

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

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

Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ ³ -4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453

Polyelectrolyte Complex Beads by Novel Two-Step Process for Improved Performance of Viable Whole-Cell Baeyer-Villiger Monoxygenase by Immobilization

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

Cited by 33 publications

References 41 publications

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

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

Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ 3 -4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453

Polyelectrolyte Complex Beads by Novel Two-Step Process for Improved Performance of Viable Whole-Cell Baeyer-Villiger Monoxygenase by Immobilization

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Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ ³ -4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453