Recent Developments in the Application of Baeyer–Villiger Monooxygenases as Biocatalysts (original) (raw)
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Baeyer–Villiger Monooxygenases, an Emerging Family of Flavin-Dependent Biocatalysts
Advanced Synthesis & Catalysis, 2003
Baeyer±Villiger monooxygenases (BVMOs) are flavoenzymes that catalyze a remarkably wide variety of oxidative reactions such as regioand enantioselective Baeyer±Villiger oxidations and sulfoxidations. Several of these conversions are difficult to achieve using chemical approaches. Due to their selectivity and catalytic efficiency, BVMOs are highly valuable biocatalysts for the synthesis of a broad range of fine chemicals. For a long time, only one member of this class of flavin-containing biocatalysts had been cloned and overexpressed which has limited their application for synthetic processes. Recently a number of new genes that encode BVMOs have been sequenced and overexpressed. In this paper the biocatalytic properties of recently cloned BVMOs are reviewed. Furthermore, the potential for obtaining novel BVMOs from sequenced genomes will be discussed.
Towards large-scale synthetic applications of Baeyer-Villiger monooxygenases
Trends in Biotechnology, 2003
Biocatalysis is coming of age, with an increasing number of reactions being scaled-up and developed. The diversity of reactions is also increasing and oxidation reactions have recently been considered for scale-up to commercial processes. One important chemical conversion, which is difficult to achieve enantio-or enantiotopo-selectively, is the Baeyer-Villiger (BV) oxidation of ketones. Using cyclohexanone monooxygenase to catalyse the reaction produces optically pure esters and lactones with exquisite enantiomeric excess values. Recently, these enzymes and their many applications in synthetic chemistry have been explored. The scale-up of these conversions has been examined with the idea of implementing the first commercial Baeyer-Villiger monooxygenase-based process. Here, we review the state-of-the-art situation for the scale-up and exploitation of these enzymes.
Applications of Baeyer-Villiger Monooxygenases in Organic Synthesis
Current Organic Chemistry, 2010
The knowledge about these Baeyer-Villiger monooxygenases has grown tremendously since the first discovery and fundamental progress in the understanding of structure, function, substrate specificities and other enzyme properties has been facilitated by the development of recombinant biocatalysts. Nature uses these biocatalysts in aerobic biodegradation pathways of cyclic and acyclic ketones and in the biosynthetic pathways of natural products. The excellent performance of Baeyer-Villiger monooxygenases in nature for the catalysis of Baeyer-Villiger oxidations with high chemo-, regio-and enantioselectivity is a role model for sustainable catalytic Baeyer-Villiger oxidations in organic synthesis. A broad range of biocatalytic conversions of cyclic ketones to lactones, linear ketones to esters, sulfoxidations and other oxidations is described. Applications in dynamic kinetic resolution as well as process and scale-up issues have been important in making this reaction platform attractive to industrial scale Baeyer-Villiger oxidations. New discoveries of Baeyer-Villiger monooxygenases in biosynthesis are promising for highly selective oxidations. and the enzymatic reaction demands a new NADPH source or a NADPH recycling system to go to completion.
A Baeyer-Villiger Oxidation Specifically Catalyzed by Human Flavin-Containing Monooxygenase 5
Drug Metabolism and Disposition, 2010
E7016, an inhibitor of poly (ADP-ribose) polymerase, is being developed for anticancer therapy. One of the major metabolites identified in preclinical animal studies was the product of an apparent oxidation and ring-opening of the 4-hydroxypiperidine. In vitro, this oxidative metabolite could not be generated by incubating E7016 with animal or human liver microsomes. Further studies revealed the formation of this unique metabolite in hepatocytes. In a NAD(P) +dependent manner, this metabolite was also generated by liver S9 fractions and recombinant human FMO5 that was fortified with liver cytosol fractions. In animal and human liver S9, this metabolic pathway could be inhibited by 4-methylpyrazole, bis-p-nitrophenylphosphate (BNPP), or a brief heat treatment at 50 ºC. Based on these results, the overall metabolic pathway was believed to involve a two-step oxidation process: dehydrogenation of the secondary alcohol in liver cytosol followed by a FMO5-mediated Baeyer-Villiger oxidation in liver microsomes. The two oxidation steps were coupled via regeneration of NAD(P) + and NAD(P)H. To further confirm this mechanism, the proposed ketone intermediate was independently synthesized. In a NAD(P)H-dependent manner, the synthetic ketone intermediate was metabolized to the same ring-opened metabolite in animal and human liver microsomes. This metabolic reaction was also inhibited by BNPP or a brief heat treatment at 50 ºC. Methimazole, the substrate/inhibitor of FMO1 and FMO3, did not inhibit this reaction. The specificity of FMO5 toward catalyzing this Baeyer-Villiger oxidation was further demonstrated by incubating the synthetic ketone intermediate in recombinant enzymes.
Multienzymatic Processes Involving Baeyer–Villiger Monooxygenases
Catalysts
Baeyer–Villiger monooxygenases (BVMOs) are flavin-dependent oxidative enzymes capable of catalyzing the insertion of an oxygen atom between a carbonylic Csp2 and the Csp3 at the alpha position, therefore transforming linear and cyclic ketones into esters and lactones. These enzymes are dependent on nicotinamides (NAD(P)H) for the flavin reduction and subsequent reaction with molecular oxygen. BVMOs can be included in cascade reactions, coupled to other redox enzymes, such as alcohol dehydrogenases (ADHs) or ene-reductases (EREDs), so that the direct conversion of alcohols or α,β-unsaturated carbonylic compounds to the corresponding esters can be achieved. In the present review, the different synthetic methodologies that have been performed by employing multienzymatic strategies with BVMOs combining whole cells or isolated enzymes, through sequential or parallel methods, are described, with the aim of highlighting the advantages of performing multienzymatic systems, and show the rece...
Selective Oxidations of Organoboron Compounds Catalyzed by Baeyer-Villiger Monooxygenases
Advanced Synthesis & Catalysis, 2011
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.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2016
Background: Ar-BVMO is a recently discovered Baeyer-Villiger monooxygenase from the genome of Acinetobacter radioresistens S13 closely related to medically relevant ethionamide monooxygenase EtaA (prodrug activator) and capable of inactivating the imipenem antibiotic. Methods: The co-substrate preference as well as steady-state and rapid kinetics studies of the recombinant purified protein were carried out using stopped-flow spectroscopy under anaerobic and aerobic conditions. K d values were measured by isothermal calorimetry. Enzymatic activity was determined by measuring the amount of product formed using high pressure liquid chromatography or gas chromatography. Site-directed mutagenesis experiments were performed to decipher the role of the active site arginine-292. Results: Ar-BVMO was found to oxidize ethionamide as well as linear ketones. Mechanistic studies on the wild type enzyme using stopped-flow spectroscopy allowed for the detection of the characteristic oxygenating C4a-(hydro)peroxyflavin intermediate, which decayed rapidly in the presence of the substrate. Replacement of arginine 292 in Ar-BVMO by glycine or alanine resulted in greatly reduced or no Baeyer-Villiger activity, respectively, demonstrating the crucial role of this residue in catalysis of ketone substrates. However, both the R292A and R292G mutants are capable of carrying out N-and S-oxidation reactions. Conclusions: Substrate profiling of Ar-BVMO confirms its close relationship to EtaA; ethionamide is one of its substrates. The active site Arginine 292 is required for its Baeyer-Villiger activity but not for heteroatom oxidation. General Significance: A single mutation converts Ar-BVMO to a unique S-or Nmonooxygenase, a useful biocatalyst for the production of oxidized metabolites of human drug metabolizing enzymes.