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Marco W. Fraaije - One of the best experts on this subject based on the ideXlab platform.
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from Thermobifida fusca
2015Co-Authors: Phenylacetone Monooxygenase, Daniel Torres E. Pazmiño, Bert-jan Baas, Dick B. Janssen, Marco W. FraaijeAbstract:The Kinetic Mechanism of PAMO from Thermobifida fusca 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-peroxy-flavin. 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. Thi
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Appl Microbiol Biotechnol (2010) 88:1135–1143 DOI 10.1007/s00253-010-2769-y BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
2013Co-Authors: Hanna M. Dudek, Daniel Torres E. Pazmiño, Gonzalo De Gonzalo, Cristina Rodríguez, Vicente Gotor, Marco W. FraaijeAbstract:Investigating the coenzyme specificity of Phenylacetone monooxygenase from Thermobifida fusc
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A stepwise approach for the reproducible optimization of PAMO expression in Escherichia coli for whole-cell biocatalysis
BMC Biotechnology, 2012Co-Authors: Edwin Van Bloois, Hanna M. Dudek, Wouter A Duetz, Marco W. FraaijeAbstract:Background Baeyer-Villiger monooxygenases (BVMOs) represent a group of enzymes of considerable biotechnological relevance as illustrated by their growing use as biocatalyst in a variety of synthetic applications. However, due to their increased use the reproducible expression of BVMOs and other biotechnologically relevant enzymes has become a pressing matter while knowledge about the factors governing their reproducible expression is scattered. Results Here, we have used Phenylacetone monooxygenase (PAMO) from Thermobifida fusca , a prototype Type I BVMO, as a model enzyme to develop a stepwise strategy to optimize the biotransformation performance of recombinant E. coli expressing PAMO in 96-well microtiter plates in a reproducible fashion. Using this system, the best expression conditions of PAMO were investigated first, including different host strains, temperature as well as time and induction period for PAMO expression. This optimized system was used next to improve biotransformation conditions, the PAMO-catalyzed conversion of Phenylacetone, by evaluating the best electron donor, substrate concentration, and the temperature and length of biotransformation. Combining all optimized parameters resulted in a more than four-fold enhancement of the biocatalytic performance and, importantly, this was highly reproducible as indicated by the relative standard deviation of 1% for non-washed cells and 3% for washed cells. Furthermore, the optimized procedure was successfully adapted for activity-based mutant screening. Conclusions Our optimized procedure, which provides a comprehensive overview of the key factors influencing the reproducible expression and performance of a biocatalyst, is expected to form a rational basis for the optimization of miniaturized biotransformations and for the design of novel activity-based screening procedures suitable for BVMOs and other NAD(P)H-dependent enzymes as well.
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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, Hugo L. Van Beek, Alessandra Pennetta, C. Martinoli, Marco W. Fraaije, 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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Blending Baeyer-Villiger monooxygenases: using a robust BVMO as a scaffold for creating chimeric enzymes with novel catalytic properties.
Chemical communications (Cambridge England), 2012Co-Authors: Hugo L. Van Beek, Gonzalo De Gonzalo, Marco W. FraaijeAbstract:The thermostable Baeyer–Villiger monooxygenase (BVMO) Phenylacetone monooxygenase (PAMO) is used as a scaffold to introduce novel selectivities from other BVMOs or the metagenome by structure-inspired subdomain exchanges. This yields biocatalysts with new preferences in the oxidation of sulfides and the Baeyer–Villiger oxidation of ketones, all while maintaining most of the original thermostability.
Robert S Phillips - One of the best experts on this subject based on the ideXlab platform.
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mutation of thermoanaerobacter ethanolicus secondary alcohol dehydrogenase at trp 110 affects stereoselectivity of aromatic ketone reduction
Organic and Biomolecular Chemistry, 2014Co-Authors: Jay M. Patel, Dewey A Sutton, Vladimir V Popik, Musa M Musa, Luis Rodriguez, Robert S PhillipsAbstract:Alcohol dehydrogenases (ADHs) are enzymes that catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. We have been studying a thermostable, nicotinamide-adenine dinucleotide phosphate (NADP+)-dependent, secondary ADH from Thermoanaerobacter ethanolicus (TeSADH). In the current work, we expanded our library of TeSADH and adopted the site-saturation mutagenesis approach in creating a comprehensive mutant library at W110. We used Phenylacetone as a model substrate to study the effectiveness of our library because this substrate showed low enantioselectivity in our previous work when reduced using W110A TeSADH. Five of the newly designed W110 mutants reduced Phenylacetone at >99.9% ee, and two of these mutants exhibit an enantiomeric ratio (E-value) of over 100. These five mutants also reduced 1-phenyl-2-butanone and 4-phenyl-2-butanone to their corresponding (S)-configured alcohols in >99.9% ee. These new mutants of TeSADH will likely have synthetic utility for reduction of aromatic ketones in the future.
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mutation of thermoanaerobacter ethanolicus secondary alcohol dehydrogenase at trp 110 affects stereoselectivity of aromatic ketone reduction
Organic and Biomolecular Chemistry, 2014Co-Authors: Jay M. Patel, Dewey A Sutton, Vladimir V Popik, Musa M Musa, Luis Rodriguez, Robert S PhillipsAbstract:Alcohol dehydrogenases (ADHs) are enzymes that catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. We have been studying a thermostable, nicotinamide-adenine dinucleotide phosphate (NADP+)-dependent, secondary ADH from Thermoanaerobacter ethanolicus (TeSADH). In the current work, we expanded our library of TeSADH and adopted the site-saturation mutagenesis approach in creating a comprehensive mutant library at W110. We used Phenylacetone as a model substrate to study the effectiveness of our library because this substrate showed low enantioselectivity in our previous work when reduced using W110A TeSADH. Five of the newly designed W110 mutants reduced Phenylacetone at >99.9% ee, and two of these mutants exhibit an enantiomeric ratio (E-value) of over 100. These five mutants also reduced 1-phenyl-2-butanone and 4-phenyl-2-butanone to their corresponding (S)-configured alcohols in >99.9% ee. These new mutants of TeSADH will likely have synthetic utility for reduction of aromatic ketones in the future.
Vicente Gotor - One of the best experts on this subject based on the ideXlab platform.
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Appl Microbiol Biotechnol (2010) 88:1135–1143 DOI 10.1007/s00253-010-2769-y BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
2013Co-Authors: Hanna M. Dudek, Daniel Torres E. Pazmiño, Gonzalo De Gonzalo, Cristina Rodríguez, Vicente Gotor, Marco W. FraaijeAbstract:Investigating the coenzyme specificity of Phenylacetone monooxygenase from Thermobifida fusc
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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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Improvement of the biocatalytic properties of one Phenylacetone monooxygenase mutant in hydrophilic organic solvents.
Enzyme and microbial technology, 2011Co-Authors: Gonzalo De Gonzalo, Cristina Rodríguez, Ana Rioz-martínez, Vicente GotorAbstract:The presence of different hydrophilic organic solvents or a water soluble polymer such as PEG 4000 led to an enhancement in the enzymatic activity of the M446G mutant of Phenylacetone monooxygenase when it is employed in enantioselective sulfoxidations and Baeyer-Villiger reactions. By solvent engineering new substrates were found to be effectively converted by this Baeyer-Villiger monooxygenase. The use of 5% methanol together with the weak anion exchange resin Lewatit MP62 also allows the dynamic kinetic resolution of a set of racemic benzylketones. By this approach (S)-benzylesters could be obtained with high yields and optical purities.
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Synthesis of Chiral 3-Alkyl-3,4-dihydroisocoumarins by Dynamic Kinetic Resolutions Catalyzed by a Baeyer—Villiger Monooxygenase.
ChemInform, 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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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.
Gonzalo De Gonzalo - One of the best experts on this subject based on the ideXlab platform.
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DOI: 10.1002/adsc.200600598 Titration and Assignment of Residues that Regulate the
2015Co-Authors: Enantioselectivity Of Phenylacetone Monooxygenase, Gonzalo De Gonzalo, Francesca Zambianchi, Marco A W. Fraaije, Giacomo B Carrea, Cristina C Rodrguez, Vicente C GotorAbstract:Abstract: Phenylacetone monooxygense (PAMO) from Thermobifida fusca was employed for the asymmetric oxidation of thioanisole (sulfooxida-tion) and of racemic 2-phenylpropionaldehyde (Baeyer–Villiger oxidation). A pH dependence of enantioselectivity was observed in both cases. Two different residues, with pKa values of 7.80.2 and 9.20.2, appeared to be responsible for the pH ef-fects on PAMO enantioselectivity. The protonation of Arg337 and the FAD:C4a-hydroperoxide/ FAD:C4a-peroxide equilibrium were identified as the major factors responsible for the fine-tuning of PAMO enantioselectivity in Baeyer–Villiger oxida-tion and sulfooxidation, respectively
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Appl Microbiol Biotechnol (2010) 88:1135–1143 DOI 10.1007/s00253-010-2769-y BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
2013Co-Authors: Hanna M. Dudek, Daniel Torres E. Pazmiño, Gonzalo De Gonzalo, Cristina Rodríguez, Vicente Gotor, Marco W. FraaijeAbstract:Investigating the coenzyme specificity of Phenylacetone monooxygenase from Thermobifida fusc
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Blending Baeyer-Villiger monooxygenases: using a robust BVMO as a scaffold for creating chimeric enzymes with novel catalytic properties.
Chemical communications (Cambridge England), 2012Co-Authors: Hugo L. Van Beek, Gonzalo De Gonzalo, Marco W. FraaijeAbstract:The thermostable Baeyer–Villiger monooxygenase (BVMO) Phenylacetone monooxygenase (PAMO) is used as a scaffold to introduce novel selectivities from other BVMOs or the metagenome by structure-inspired subdomain exchanges. This yields biocatalysts with new preferences in the oxidation of sulfides and the Baeyer–Villiger oxidation of ketones, all while maintaining most of the original thermostability.
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Blending Baeyer–Villiger monooxygenases: using a robust BVMO as a scaffold for creating chimeric enzymes with novel catalytic properties
2012Co-Authors: Beek, Hugo L. Van, Gonzalo De Gonzalo, Fraaije, Marco W.Abstract:The thermostable Baeyer–Villiger monooxygenase (BVMO) Phenylacetone monooxygenase (PAMO) is used as a scaffold to introduce novel selectivities from other BVMOs or the metagenome by structure-inspired subdomain exchanges. This yields biocatalysts with new preferences in the oxidation of sulfides and the Baeyer–Villiger oxidation of ketones, all while maintaining most of the original thermostability.
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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).
Jay M. Patel - One of the best experts on this subject based on the ideXlab platform.
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mutation of thermoanaerobacter ethanolicus secondary alcohol dehydrogenase at trp 110 affects stereoselectivity of aromatic ketone reduction
Organic and Biomolecular Chemistry, 2014Co-Authors: Jay M. Patel, Dewey A Sutton, Vladimir V Popik, Musa M Musa, Luis Rodriguez, Robert S PhillipsAbstract:Alcohol dehydrogenases (ADHs) are enzymes that catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. We have been studying a thermostable, nicotinamide-adenine dinucleotide phosphate (NADP+)-dependent, secondary ADH from Thermoanaerobacter ethanolicus (TeSADH). In the current work, we expanded our library of TeSADH and adopted the site-saturation mutagenesis approach in creating a comprehensive mutant library at W110. We used Phenylacetone as a model substrate to study the effectiveness of our library because this substrate showed low enantioselectivity in our previous work when reduced using W110A TeSADH. Five of the newly designed W110 mutants reduced Phenylacetone at >99.9% ee, and two of these mutants exhibit an enantiomeric ratio (E-value) of over 100. These five mutants also reduced 1-phenyl-2-butanone and 4-phenyl-2-butanone to their corresponding (S)-configured alcohols in >99.9% ee. These new mutants of TeSADH will likely have synthetic utility for reduction of aromatic ketones in the future.
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mutation of thermoanaerobacter ethanolicus secondary alcohol dehydrogenase at trp 110 affects stereoselectivity of aromatic ketone reduction
Organic and Biomolecular Chemistry, 2014Co-Authors: Jay M. Patel, Dewey A Sutton, Vladimir V Popik, Musa M Musa, Luis Rodriguez, Robert S PhillipsAbstract:Alcohol dehydrogenases (ADHs) are enzymes that catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. We have been studying a thermostable, nicotinamide-adenine dinucleotide phosphate (NADP+)-dependent, secondary ADH from Thermoanaerobacter ethanolicus (TeSADH). In the current work, we expanded our library of TeSADH and adopted the site-saturation mutagenesis approach in creating a comprehensive mutant library at W110. We used Phenylacetone as a model substrate to study the effectiveness of our library because this substrate showed low enantioselectivity in our previous work when reduced using W110A TeSADH. Five of the newly designed W110 mutants reduced Phenylacetone at >99.9% ee, and two of these mutants exhibit an enantiomeric ratio (E-value) of over 100. These five mutants also reduced 1-phenyl-2-butanone and 4-phenyl-2-butanone to their corresponding (S)-configured alcohols in >99.9% ee. These new mutants of TeSADH will likely have synthetic utility for reduction of aromatic ketones in the future.
