6 results on '"Dudek, Hanna M."'
Search Results
2. A stepwise approach for the reproducible optimization of PAMO expression in Escherichia coli for whole-cell biocatalysis
- Author
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van Bloois Edwin, Dudek Hanna M, Duetz Wouter A, and Fraaije Marco W
- Subjects
Baeyer-Villiger monoxygenase ,Escherichia coli ,Biocatalysis ,Square deep-well microtiter plates ,Screening ,Biotechnology ,TP248.13-248.65 - Abstract
Abstract 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.
- Published
- 2012
- Full Text
- View/download PDF
3. Mapping the Substrate Binding Site of Phenylacetone Monooxygenase from Thermobifida fusca by Mutational Analysis
- Author
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Dudek, Hanna M., Gonzalo Calvo , de, Gonzalo, Torres Pazmino, Daniel, Stepniak, Piotr, Wyrwicz, Lucjan S., Rychlewski, Leszek, Fraaije, Marco W., Stępniak, Piotr, and Biotechnology
- Subjects
Models, Molecular ,Stereochemistry ,DIRECTED EVOLUTION ,DNA Mutational Analysis ,Molecular Sequence Data ,ENANTIOSELECTIVITY ,Biology ,Applied Microbiology and Biotechnology ,SEQUENCE ,Mixed Function Oxygenases ,Substrate Specificity ,Acetone ,CLONING ,Cyclopentanone monooxygenase ,Actinomycetales ,Enzyme Stability ,Escherichia coli ,Amino Acid Sequence ,Enzymology and Protein Engineering ,Binding site ,SPECIFICITY ,Phenylacetone monooxygenase ,Thermostability ,Binding Sites ,Sequence Homology, Amino Acid ,Ecology ,Active site ,Monooxygenase ,Directed evolution ,CYCLOHEXANONE MONOOXYGENASES ,Biochemistry ,Genes, Bacterial ,Mutation ,Mutagenesis, Site-Directed ,Oxygenases ,biology.protein ,BAEYER-VILLIGER MONOOXYGENASES ,KINETIC RESOLUTION ,Oxidation-Reduction ,Sequence Alignment ,Algorithms ,Protein Binding ,Food Science ,Biotechnology ,BIOCATALYSTS - Abstract
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.
- Published
- 2011
4. Redesign of Baeyer–Villiger Monooxygenases for Synthetic Applications
- Author
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Dudek, Hanna M., Fraaije, Marco, Biotechnology, and Groningen Biomolecular Sciences and Biotechnology
- Subjects
Fenylenonen ,NADPH ,Oxygenases ,Proefschriften (vorm) ,Protein engineering ,Vormgeving ,enzymen, hormonen, vitaminen (biochemie) ,Biokatalyse - Abstract
Herontwerpen van Baeyer–Villiger monooxygenases voor synthetische applicaties (korte samenvatting) In het veld van biokatalyse worden enzymen toegepast om chemische reacties te katalyseren, die kunnen leiden tot efficiëntere en milieuvriendelijkere processen. Tegenwoordig is het mogelijk om eigenschappen van enzymen aan te passen met behulp van “protein engineering” methodes. Het onderzoek dat beschreven wordt in dit proefschrift, focust zich voornamelijk op het modificeren van fenylaceton monooxygenase (PAMO), een enzym dat behoort tot de Baeyer–Villiger monooxygenase familie, tot een efficiëntere biokatalyst. Allereerst, is er geprobeerd om de coenzym en substraatspecificiteit aan te passen door plaats-gerichte mutagenese. Dit resulteerde in gematigde verbeteringen van de enzymeigenschappen, maar niettemin verbeterde het onze kennis over dit type enzymen. Door het beperkte succes van onze experimenten, hebben we de focus verlegd naar alternatieve methodes en hebben we gekozen voor het ontwikkelen van een activiteitsassay om mutant collecties te kunnen screenen. Deze nieuwe activiteitsassay is gebaseerd op het gebruik van een tweede enzym dat gekoppeld is aan de activiteit van PAMO en kon worden toegepast op het screenen van een grote hoeveelheid enzym varianten. Met behulp van gedetailleerde kennis over het actieve centrum van het enzym, het katalytisch mechanisme dat voortkomt uit structurele studies, modeleren, kinetische analyses en karakterisatie van varianten is het mogelijk geworden om banken te ontwerpen van hoge kwaliteit, terwijl efficiënte screeningsmethodes het mogelijk maken om verbeterde varianten te herkennen. Deze aanpak is succesvol gebleken voor PAMO en kan worden toegepast in het herontwerpen of aanpassen van andere (Baeyer–Villiger) monooxygenases, mede doordat de ontwikkelde screeningsmethode generiek is voor NADPH-verbruikende enzymen.
- Published
- 2013
5. Snapshots of Enzymatic Baeyer-Villiger Catalysis OXYGEN ACTIVATION AND INTERMEDIATE STABILIZATION
- Author
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Orru, Roberto, Dudek, Hanna M., Martinoli, Christian, Torres Pazmiño, Daniel E., Royant, Antoine, Weik, Martin, Fraaije, Marco W., Mattevi, Andrea, Groningen Biomolecular Sciences and Biotechnology, and Biotechnology
- Subjects
MECHANISM ,PHENYLACETONE-MONOOXYGENASE ,THERMOBIFIDA-FUSCA ,FLAVIN-CONTAINING MONOOXYGENASE ,CRYSTALLOGRAPHY ,CYCLOHEXANONE MONOOXYGENASE ,Catalysis ,Mixed Function Oxygenases ,Acetone ,Oxygen ,CRYSTALS ,Bacterial Proteins ,Models, Chemical ,Actinomycetales ,Enzymology ,SPECIFICITY ,OXIDATIONS ,NADP ,BIOCATALYSTS - Abstract
Baeyer-Villiger monooxygenases catalyze the oxidation of carbonylic substrates to ester or lactone products using NADPH as electron donor and molecular oxygen as oxidative reactant. Using protein engineering, kinetics, microspectrophotometry, crystallography, and intermediate analogs, we have captured several snapshots along the catalytic cycle which highlight key features in enzyme catalysis. After acting as electron donor, the enzyme-bound NADP(H) forms an H-bond with the flavin cofactor. This interaction is critical for stabilizing the oxygen-activating flavin-peroxide intermediate that results from the reaction of the reduced cofactor with oxygen. An essential active-site arginine acts as anchoring element for proper binding of the ketone substrate. Its positively charged guanidinium group can enhance the propensity of the substrate to undergo a nucleophilic attack by the flavin-peroxide intermediate. Furthermore, the arginine side chain, together with the NADP(+) ribose group, forms the niche that hosts the negatively charged Criegee intermediate that is generated upon reaction of the substrate with the flavin-peroxide. The fascinating ability of Baeyer-Villiger monooxygenases to catalyze a complex multistep catalytic reaction originates from concerted action of this Arg-NADP(H) pair and the flavin subsequently to promote flavin reduction, oxygen activation, tetrahedral intermediate formation, and product synthesis and release. The emerging picture is that these enzymes are mainly oxygen-activating and "Criegee-stabilizing" catalysts that act on any chemically suitable substrate that can diffuse into the active site, emphasizing their potential value as toolboxes for biocatalytic applications.
- Published
- 2011
6. Snapshots of enzymatic Baeyer-Villiger catalysis: oxygen activation and intermediate stabilization.
- Author
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Orru R, Dudek HM, Martinoli C, Torres Pazmiño DE, Royant A, Weik M, Fraaije MW, and Mattevi A
- Subjects
- Acetone analogs & derivatives, Acetone chemistry, Catalysis, Actinomycetales enzymology, Bacterial Proteins chemistry, Mixed Function Oxygenases chemistry, Models, Chemical, NADP chemistry, Oxygen chemistry
- Abstract
Baeyer-Villiger monooxygenases catalyze the oxidation of carbonylic substrates to ester or lactone products using NADPH as electron donor and molecular oxygen as oxidative reactant. Using protein engineering, kinetics, microspectrophotometry, crystallography, and intermediate analogs, we have captured several snapshots along the catalytic cycle which highlight key features in enzyme catalysis. After acting as electron donor, the enzyme-bound NADP(H) forms an H-bond with the flavin cofactor. This interaction is critical for stabilizing the oxygen-activating flavin-peroxide intermediate that results from the reaction of the reduced cofactor with oxygen. An essential active-site arginine acts as anchoring element for proper binding of the ketone substrate. Its positively charged guanidinium group can enhance the propensity of the substrate to undergo a nucleophilic attack by the flavin-peroxide intermediate. Furthermore, the arginine side chain, together with the NADP(+) ribose group, forms the niche that hosts the negatively charged Criegee intermediate that is generated upon reaction of the substrate with the flavin-peroxide. The fascinating ability of Baeyer-Villiger monooxygenases to catalyze a complex multistep catalytic reaction originates from concerted action of this Arg-NADP(H) pair and the flavin subsequently to promote flavin reduction, oxygen activation, tetrahedral intermediate formation, and product synthesis and release. The emerging picture is that these enzymes are mainly oxygen-activating and "Criegee-stabilizing" catalysts that act on any chemically suitable substrate that can diffuse into the active site, emphasizing their potential value as toolboxes for biocatalytic applications.
- Published
- 2011
- Full Text
- View/download PDF
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