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Marco W. Fraaije - One of the best experts on this subject based on the ideXlab platform.
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Side-Chain Pruning Has Limited Impact on Substrate Preference in a Promiscuous Enzyme
2018Co-Authors: Maximilian J. L. J. Fürst, Marco W. Fraaije, Elvira Romero, Rúben Gómez J. Castellanos, Andrea MatteviAbstract:Detoxifying enzymes such as flavin-containing Monooxygenases deal with a huge array of highly diverse xenobiotics and toxic compounds. In addition to being of high physiological relevance, these drug-metabolizing enzymes are useful catalysts for synthetic chemistry. Despite the wealth of studies, the molecular basis of their relaxed substrate selectivity remains an open question. Here, we addressed this issue by applying a cumulative alanine mutagenesis approach to cyclohexanone Monooxygenase from Thermocrispum municipale, a flavin-dependent Baeyer–Villiger Monooxygenase which we chose as a model system because of its pronounced thermostability and substrate promiscuity. Simultaneous removal of up to eight noncatalytic active-site side chains including four phenylalanines had no effect on protein folding, thermostability, and cofactor loading. We observed a linear decrease in activity, rather than a selectivity switch, and attributed this to a less efficient catalytic environment in the enlarged active-site space. Time-resolved kinetic studies confirmed this interpretation. We also determined the crystal structure of the enzyme in complex with a mimic of the reaction intermediate that shows an unaltered overall protein conformation. These findings led us to propose that this cyclohexanone Monooxygenase may lack a distinct substrate selection mechanism altogether. We speculate that the main or exclusive function of the protein shell in promiscuous enzymes might be the stabilization and accessibility of their very reactive catalytic intermediates
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Exploring the Structural Basis of Substrate Preferences in Baeyer-Villiger Monooxygenases INSIGHT FROM STEROID Monooxygenase
Journal of Biological Chemistry, 2012Co-Authors: Stefano Franceschini, Marco W. Fraaije, Hugo L. Van Beek, Alessandra Pennetta, C. Martinoli, Andrea MatteviAbstract:Abstract Steroid Monooxygenase (STMO) from Rhodococcus rhodochrous catalyzes the Baeyer-Villiger conversion of progesterone into progesterone acetate using FAD as prosthetic group and NADPH as reducing cofactor. The enzyme shares high sequence similarity with well characterized Baeyer-Villiger Monooxygenases, including phenylacetone Monooxygenase and cyclohexanone Monooxygenase. The comparative biochemical and structural analysis of STMO can be particularly insightful with regard to the understanding of the substrate-specificity properties of Baeyer-Villiger Monooxygenases that are emerging as promising tools in biocatalytic applications and as targets for prodrug activation. The crystal structures of STMO in the native, NADP+-bound, and two mutant forms reveal structural details on this microbial steroid-degrading enzyme. The binding of the nicotinamide ring of NADP+ is shifted with respect to the flavin compared with that observed in other Monooxygenases of the same class. This finding fully supports the idea that NADP(H) adopts various positions during the catalytic cycle to perform its multiple functions in catalysis. The active site closely resembles that of phenylacetone Monooxygenase. This observation led us to discover that STMO is capable of acting also on phenylacetone, which implies an impressive level of substrate promiscuity. The investigation of six mutants that target residues on the surface of the substrate-binding site reveals that enzymatic conversions of both progesterone and phenylacetone are largely insensitive to relatively drastic amino acid changes, with some mutants even displaying enhanced activity on progesterone. These features possibly reflect the fact that these enzymes are continuously evolving to acquire new activities, depending on the emerging availabilities of new compounds in the living environment.
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mapping the substrate binding site of phenylacetone Monooxygenase from thermobifida fusca by mutational analysis
Applied and Environmental Microbiology, 2011Co-Authors: Hanna M Dudek, Daniel Torres E. Pazmiño, Gonzalo De Gonzalo, Piotr Stepniak, Lucjan Wyrwicz, Leszek Rychlewski, Marco W. FraaijeAbstract: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.
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Synthesis of chiral 3-alkyl-3,4-dihydroisocoumarins by dynamic kinetic resolutions catalyzed by a Baeyer-Villiger Monooxygenase.
The Journal of organic chemistry, 2010Co-Authors: Ana Rioz-martínez, Marco W. Fraaije, Daniel Torres E. Pazmiño, Gonzalo De Gonzalo, Vicente GotorAbstract:Baeyer−Villiger Monooxygenases have been tested in the oxidation of racemic benzofused ketones. When employing a single mutant of phenylacetone Monooxygenase (M446G PAMO) under the proper reaction conditions, it was possible to achieve 3-substituted 3,4-dihydroisocoumarins with high yields and optical purities through regioselective dynamic kinetic resolution processes.
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multiple pathways guide oxygen diffusion into flavoenzyme active sites
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Riccardo Baron, Willem J H Van Berkel, Conor T Riley, Pirom Chenprakhon, Kittisak Thotsaporn, Remko T Winter, Andrea Alfieri, Federico Forneris, Pimchai Chaiyen, Marco W. FraaijeAbstract:Dioxygen (O(2)) and other gas molecules have a fundamental role in a variety of enzymatic reactions. However, it is only poorly understood which O(2) uptake mechanism enzymes employ to promote efficient catalysis and how general this is. We investigated O(2) diffusion pathways into Monooxygenase and oxidase flavoenzymes, using an integrated computational and experimental approach. Enhanced-statistics molecular dynamics simulations reveal spontaneous protein-guided O(2) diffusion from the bulk solvent to preorganized protein cavities. The predicted protein-guided diffusion paths and the importance of key cavity residues for oxygen diffusion were verified by combining site-directed mutagenesis, rapid kinetics experiments, and high-resolution X-ray structures. This study indicates that Monooxygenase and oxidase flavoenzymes employ multiple funnel-shaped diffusion pathways to absorb O(2) from the solvent and direct it to the reacting C4a atom of the flavin cofactor. The difference in O(2) reactivity among dehydrogenases, Monooxygenases, and oxidases ultimately resides in the fine modulation of the local environment embedding the reactive locus of the flavin.
Dick B Janssen - One of the best experts on this subject based on the ideXlab platform.
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Kinetic Mechanism of Phenylacetone Monooxygenase from Thermobifida fusca
Biochemistry, 2008Co-Authors: Daniel Torres E. Pazmiño, Dick B Janssen, Bert-jan Baas, Marco W. FraaijeAbstract:Phenylacetone Monooxygenase (PAMO) from Thermobifida fusca is a FAD-containing Baeyer-Villiger Monooxygenase (BVMO). To elucidate the mechanism of conversion of phenylacetone by PAMO, we have performed a detailed steady-state and pre-steady-state kinetic analysis. In the catalytic cycle ( k cat = 3.1 s (-1)), rapid binding of NADPH ( K d = 0.7 microM) is followed by a transfer of the 4( R)-hydride from NADPH to the FAD cofactor ( k red = 12 s (-1)). The reduced PAMO is rapidly oxygenated by molecular oxygen ( k ox = 870 mM (-1) s (-1)), yielding a C4a-peroxyflavin. The peroxyflavin enzyme intermediate reacts with phenylacetone to form benzylacetate ( k 1 = 73 s (-1)). This latter kinetic event leads to an enzyme intermediate which we could not unequivocally assign and may represent a Criegee intermediate or a C4a-hydroxyflavin form. The relatively slow decay (4.1 s (-1)) of this intermediate yields fully reoxidized PAMO and limits the turnover rate. NADP (+) release is relatively fast and represents the final step of the catalytic cycle. This study shows that kinetic behavior of PAMO is significantly different when compared with that of sequence-related Monooxygenases, e.g., cyclohexanone Monooxygenase and liver microsomal flavin-containing Monooxygenase. Inspection of the crystal structure of PAMO has revealed that residue R337, which is conserved in other BVMOs, is positioned close to the flavin cofactor. The analyzed R337A and R337K mutant enzymes were still able to form and stabilize the C4a-peroxyflavin intermediate. The mutants were unable to convert either phenylacetone or benzyl methyl sulfide. This demonstrates that R337 is crucially involved in assisting PAMO-mediated Baeyer-Villiger and sulfoxidation reactions.
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Kinetic Mechanism of Phenylacetone Monooxygenase from Thermobifida fusca †
2007Co-Authors: Daniel Torres E. Pazmiño, Dick B Janssen, Bert-jan Baas, Marco W. FraaijeAbstract:ABSTRACT: Phenylacetone Monooxygenase (PAMO) from Thermobifida fusca is a FAD-containing Baeyer-Villiger Monooxygenase (BVMO). To elucidate the mechanism of conversion of phenylacetone by PAMO, we have performed a detailed steady-state and pre-steady-state kinetic analysis. In the catalytic cycle (kcat) 3.1 s-1), rapid binding of NADPH (Kd) 0.7 µM) is followed by a transfer of the 4(R)-hydride from NADPH to the FAD cofactor (kred) 12 s-1). The reduced PAMO is rapidly oxygenated by molecular oxygen (kox) 870 mM-1 s-1), yielding a C4a-peroxyflavin. The peroxyflavin enzyme intermediate reacts with phenylacetone to form benzylacetate (k1) 73 s-1). This latter kinetic event leads to an enzyme intermediate which we could not unequivocally assign and may represent a Criegee intermediate or a C4a-hydroxyflavin form. The relatively slow decay (4.1 s-1) of this intermediate yields fully reoxidized PAMO and limits the turnover rate. NADP + release is relatively fast and represents the final step of the catalytic cycle. This study shows that kinetic behavior of PAMO is significantly different when compared with that of sequence-related Monooxygenases, e.g., cyclohexanone Monooxygenase and liver microsomal flavin-containing Monooxygenase. Inspection of the crystal structure of PAMO has revealed that residue R337, which is conserved in other BVMOs, is positioned close to the flavin cofactor. The analyzed R337A and R337K mutant enzymes were still able to form and stabilize the C4a-peroxyflavin intermediate. The mutants were unable to convert either phenylacetone or benzyl methyl sulfide. This demonstrates tha
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discovery of a thermostable baeyer villiger Monooxygenase by genome mining
Applied Microbiology and Biotechnology, 2005Co-Authors: Marco W. Fraaije, Dominic P H M Heuts, Erik W Van Hellemond, Jeffrey Lutje H Spelberg, Dick B JanssenAbstract:Baeyer–Villiger Monooxygenases represent useful biocatalytic tools, as they can catalyze reactions which are difficult to achieve using chemical means. However, only a limited number of these atypical Monooxygenases are available in recombinant form. Using a recently described protein sequence motif, a putative Baeyer–Villiger Monooxygenase (BVMO) was identified in the genome of the thermophilic actinomycete Thermobifida fusca. Heterologous expression of the respective protein in Escherichia coli and subsequent enzyme characterization showed that it indeed represents a BVMO. The NADPH-dependent and FAD-containing Monooxygenase is active with a wide range of aromatic ketones, while aliphatic substrates are also converted. The best substrate discovered so far is phenylacetone (kcat = 1.9 s−1, KM = 59 μM). The enzyme exhibits moderate enantioselectivity with α-methylphenylacetone (enantiomeric ratio of 7). In addition to Baeyer–Villiger reactions, the enzyme is able to perform sulfur oxidations. Different from all known BVMOs, this newly identified biocatalyst is relatively thermostable, displaying an activity half-life of 1 day at 52°C. This study demonstrates that, using effective annotation tools, genomes can efficiently be exploited as a source of novel BVMOs.
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identification of a baeyer villiger Monooxygenase sequence motif
FEBS Letters, 2002Co-Authors: Marco W. Fraaije, Nanne M. Kamerbeek, Willem J H Van Berkel, Dick B JanssenAbstract:Baeyer–Villiger Monooxygenases (BVMOs) form a distinct class of flavoproteins that catalyze the insertion of an oxygen atom in a C–C bond using dioxygen and NAD(P)H. Using newly characterized BVMO sequences, we have uncovered a BVMO-identifying sequence motif: FXGXXXHXXXW(P/D). Studies with site-directed mutants of 4-hydroxyacetophenone Monooxygenase from Pseudomonas fluorescens ACB suggest that this fingerprint sequence is critically involved in catalysis. Further sequence analysis showed that the BVMOs belong to a novel superfamily that comprises three known classes of FAD-dependent Monooxygenases: the so-called flavin-containing Monooxygenases (FMOs), the N-hydroxylating Monooxygenases (NMOs), and the BVMOs. Interestingly, FMOs contain an almost identical sequence motif when compared to the BVMO sequences: FXGXXXHXXX(Y/F). Using these novel amino acid sequence fingerprints, BVMOs and FMOs can be readily identified in the protein sequence databank.
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4 hydroxyacetophenone Monooxygenase from pseudomonas fluorescens acb a novel flavoprotein catalyzing baeyer villiger oxidation of aromatic compounds
FEBS Journal, 2001Co-Authors: Nanne M. Kamerbeek, Willem J. H. Van Berkel, Marco W. Fraaije, Maria Lle J H Moonen, Dick B JanssenAbstract:A novel flavoprotein that catalyses the NADPH-dependent oxidation of 4-hydroxyacetophenone to 4-hydroxyphenyl acetate, was purified to homogeneity from Pseudomonas fluorescens ACB. Characterization of the purified enzyme showed that 4-hydroxyacetophenone Monooxygenase (HAPMO) is a homodimer of approximate to 140 kDa with each subunit containing a noncovalently bound FAD molecule. HAPMO displays a tight coupling between NADPH oxidation and substrate oxygenation. Besides 4-hydroxyacetophenone a wide range of other acetophenones are readily converted via a Baeyer-Villiger rearrangement reaction into the corresponding phenyl acetates. The P. fluorescens HAPMO gene (hapE) was characterized. It encoded a 640 amino-acid protein with a deduced mass of 71 884 Da. Except for an N-terminal extension of approximate to 135 residues, the sequence of HAPMO shares significant similarity with two known types of Baeyer-Villiger Monooxygenases: cyclohexanone Monooxygenase (27-33% sequence identity) and steroid Monooxygenase (33% sequence identity). The HAPMO sequence contains several sequence motifs indicative for the presence of two Rossman fold domains involved in FAD and NADPH binding. The functional role of a recently identified flavoprotein sequence motif (ATG) was explored by site-directed mutagenesis. Replacement of the strictly conserved glycine (G490) resulted in a dramatic effect on catalysis. From a kinetic analysis of the G490A mutant it is concluded that the observed sequence motif serves a structural function which is of importance for NADPH binding.
Gonzalo De Gonzalo - One of the best experts on this subject based on the ideXlab platform.
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Optimization of oxidative bioconversions catalyzed by phenylacetone Monooxygenase from Thermobifida fusca
Journal of Molecular Catalysis B-enzymatic, 2011Co-Authors: Cristina Rodríguez, Gonzalo De Gonzalo, Vicente GotorAbstract:Abstract By choosing properly the nature of the reaction medium and its ionic strength, biocatalytic properties of isolated phenylacetone Monooxygenase from Thermobifida fusca can be improved, achieving the best results when working in Tris or phosphate buffers presenting moderate ionic strengths. The use of different enzymatic cofactor regenerating systems has been studied, resulting in the highest activities by using glucose or glucose-6-phosphate dehydrogenase. The cofactor concentration, key parameter when oxidizing with isolated Baeyer–Villiger Monooxygenases, was optimized, being demonstrated that PAMO can perform its biocatalytic activity with the highest TTNs with low requirement of nicotinamide cofactor (2 μM).
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mapping the substrate binding site of phenylacetone Monooxygenase from thermobifida fusca by mutational analysis
Applied and Environmental Microbiology, 2011Co-Authors: Hanna M Dudek, Daniel Torres E. Pazmiño, Gonzalo De Gonzalo, Piotr Stepniak, Lucjan Wyrwicz, Leszek Rychlewski, Marco W. FraaijeAbstract: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.
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Selective Oxidations of Organoboron Compounds Catalyzed by Baeyer–Villiger Monooxygenases
2011Co-Authors: Brondani, Patrícia B., Gonzalo De Gonzalo, Fraaije, Marco W., Andrade, Leandro H.Abstract:The applicability of Baeyer–Villiger Monooxygenases (BVMOs) in organoboron chemistry has been explored through testing chemo- and enantioselective oxidations of a variety of boron-containing aromatic and vinylic compounds. Several BVMOs, namely: phenylacetone Monooxygenase (PAMO), M446G PAMO mutant, 4-hydroxyacetophenone Monooxygenase (HAPMO) and cyclohexanone Monooxygenase (CHMO) were used in this study. The degree of chemoselectivity depends on the type of BVMO employed, in which the biocatalysts prefer boron-carbon oxidation over Baeyer–Villiger oxidation or epoxidation. Interestingly, it was discovered that PAMO can be used to perform kinetic resolution of boron-containing compounds with good enantioselectivities. These findings extend the known biocatalytic repertoire of BVMOs by showing a new family of compounds that can be oxidized by these enzymes.
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Selective Oxidations of Organoboron Compounds Catalyzed by Baeyer-Villiger Monooxygenases
WILEY-BLACKWELL, 2011Co-Authors: Brondani, Patrícia B., Gonzalo De Gonzalo, Fraaije, Marco W., Andrade, Leandro H.Abstract:The applicability of Baeyer-Villiger Monooxygenases (BVMOs) in organoboron chemistry has been explored through testing chemo-and enantioselective oxidations of a variety of boron-containing aromatic and vinylic compounds. Several BVMOs, namely: phenylacetone Monooxygenase (PAMO), M446G PAMO mutant, 4-hydroxyacetophenone Monooxygenase (HAPMO) and cyclohexanone Monooxygenase (CHMO) were used in this study. The degree of chemoselectivity depends on the type of BVMO employed, in which the biocatalysts prefer boron-carbon oxidation over Baeyer-Villiger oxidation or epoxidation. Interestingly, it was discovered that PAMO can be used to perform kinetic resolution of boron-containing compounds with good enantioselectivities. These findings extend the known biocatalytic repertoire of BVMOs by showing a new family of compounds that can be oxidized by these enzymes
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Selective Oxidations of Organoboron Compounds Catalyzed by Baeyer-Villiger Monooxygenases
WILEY-BLACKWELL, 2011Co-Authors: Brondani, Patrícia B., Gonzalo De Gonzalo, Fraaije, Marco W., Andrade, Leandro H.Abstract:The applicability of Baeyer-Villiger Monooxygenases (BVMOs) in organoboron chemistry has been explored through testing chemo-and enantioselective oxidations of a variety of boron-containing aromatic and vinylic compounds. Several BVMOs, namely: phenylacetone Monooxygenase (PAMO), M446G PAMO mutant, 4-hydroxyacetophenone Monooxygenase (HAPMO) and cyclohexanone Monooxygenase (CHMO) were used in this study. The degree of chemoselectivity depends on the type of BVMO employed, in which the biocatalysts prefer boron-carbon oxidation over Baeyer-Villiger oxidation or epoxidation. Interestingly, it was discovered that PAMO can be used to perform kinetic resolution of boron-containing compounds with good enantioselectivities. These findings extend the known biocatalytic repertoire of BVMOs by showing a new family of compounds that can be oxidized by these enzymes.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPqCAPESCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FAPESPFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)EUE
Vicente Gotor - One of the best experts on this subject based on the ideXlab platform.
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Optimization of oxidative bioconversions catalyzed by phenylacetone Monooxygenase from Thermobifida fusca
Journal of Molecular Catalysis B-enzymatic, 2011Co-Authors: Cristina Rodríguez, Gonzalo De Gonzalo, Vicente GotorAbstract:Abstract By choosing properly the nature of the reaction medium and its ionic strength, biocatalytic properties of isolated phenylacetone Monooxygenase from Thermobifida fusca can be improved, achieving the best results when working in Tris or phosphate buffers presenting moderate ionic strengths. The use of different enzymatic cofactor regenerating systems has been studied, resulting in the highest activities by using glucose or glucose-6-phosphate dehydrogenase. The cofactor concentration, key parameter when oxidizing with isolated Baeyer–Villiger Monooxygenases, was optimized, being demonstrated that PAMO can perform its biocatalytic activity with the highest TTNs with low requirement of nicotinamide cofactor (2 μM).
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Synthesis of chiral 3-alkyl-3,4-dihydroisocoumarins by dynamic kinetic resolutions catalyzed by a Baeyer-Villiger Monooxygenase.
The Journal of organic chemistry, 2010Co-Authors: Ana Rioz-martínez, Marco W. Fraaije, Daniel Torres E. Pazmiño, Gonzalo De Gonzalo, Vicente GotorAbstract:Baeyer−Villiger Monooxygenases have been tested in the oxidation of racemic benzofused ketones. When employing a single mutant of phenylacetone Monooxygenase (M446G PAMO) under the proper reaction conditions, it was possible to achieve 3-substituted 3,4-dihydroisocoumarins with high yields and optical purities through regioselective dynamic kinetic resolution processes.
Nanne M. Kamerbeek - One of the best experts on this subject based on the ideXlab platform.
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Flavoprotein Monooxygenases, a diverse class of oxidative biocatalysts.
Journal of Biotechnology, 2006Co-Authors: Willem J. H. Van Berkel, Nanne M. Kamerbeek, Marco W. FraaijeAbstract:During the last decades a large number of flavin-dependent Monooxygenases have been isolated and studied. This has revealed that flavoprotein Monooxygenases are able to catalyze a remarkable wide variety of oxidative reactions such as regioselective hydroxylations and enantioselective sulfoxidations. These oxidation reactions are often difficult, if not impossible, to be achieved using chemical approaches. Analysis of the available genome sequences has indicated that many more flavoprotein Monooxygenases exist and await biocatalytic exploration. Based on the known biochemical properties of a number of flavoprotein Monooxygenases and sequence and structural analyses, flavoprotein Monooxygenases can be classified into six distinct flavoprotein Monooxygenase subclasses. This review provides an inventory of known flavoprotein Monooxygenases belonging to these different enzyme subclasses. Furthermore, the biocatalytic potential of a selected number of flavoprotein Monooxygenases is highlighted.
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identification of a baeyer villiger Monooxygenase sequence motif
FEBS Letters, 2002Co-Authors: Marco W. Fraaije, Nanne M. Kamerbeek, Willem J H Van Berkel, Dick B JanssenAbstract:Baeyer–Villiger Monooxygenases (BVMOs) form a distinct class of flavoproteins that catalyze the insertion of an oxygen atom in a C–C bond using dioxygen and NAD(P)H. Using newly characterized BVMO sequences, we have uncovered a BVMO-identifying sequence motif: FXGXXXHXXXW(P/D). Studies with site-directed mutants of 4-hydroxyacetophenone Monooxygenase from Pseudomonas fluorescens ACB suggest that this fingerprint sequence is critically involved in catalysis. Further sequence analysis showed that the BVMOs belong to a novel superfamily that comprises three known classes of FAD-dependent Monooxygenases: the so-called flavin-containing Monooxygenases (FMOs), the N-hydroxylating Monooxygenases (NMOs), and the BVMOs. Interestingly, FMOs contain an almost identical sequence motif when compared to the BVMO sequences: FXGXXXHXXX(Y/F). Using these novel amino acid sequence fingerprints, BVMOs and FMOs can be readily identified in the protein sequence databank.
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4 hydroxyacetophenone Monooxygenase from pseudomonas fluorescens acb a novel flavoprotein catalyzing baeyer villiger oxidation of aromatic compounds
FEBS Journal, 2001Co-Authors: Nanne M. Kamerbeek, Willem J. H. Van Berkel, Marco W. Fraaije, Maria Lle J H Moonen, Dick B JanssenAbstract:A novel flavoprotein that catalyses the NADPH-dependent oxidation of 4-hydroxyacetophenone to 4-hydroxyphenyl acetate, was purified to homogeneity from Pseudomonas fluorescens ACB. Characterization of the purified enzyme showed that 4-hydroxyacetophenone Monooxygenase (HAPMO) is a homodimer of approximate to 140 kDa with each subunit containing a noncovalently bound FAD molecule. HAPMO displays a tight coupling between NADPH oxidation and substrate oxygenation. Besides 4-hydroxyacetophenone a wide range of other acetophenones are readily converted via a Baeyer-Villiger rearrangement reaction into the corresponding phenyl acetates. The P. fluorescens HAPMO gene (hapE) was characterized. It encoded a 640 amino-acid protein with a deduced mass of 71 884 Da. Except for an N-terminal extension of approximate to 135 residues, the sequence of HAPMO shares significant similarity with two known types of Baeyer-Villiger Monooxygenases: cyclohexanone Monooxygenase (27-33% sequence identity) and steroid Monooxygenase (33% sequence identity). The HAPMO sequence contains several sequence motifs indicative for the presence of two Rossman fold domains involved in FAD and NADPH binding. The functional role of a recently identified flavoprotein sequence motif (ATG) was explored by site-directed mutagenesis. Replacement of the strictly conserved glycine (G490) resulted in a dramatic effect on catalysis. From a kinetic analysis of the G490A mutant it is concluded that the observed sequence motif serves a structural function which is of importance for NADPH binding.
