Characterization of a new Baeyer-Villiger monooxygenase and conversion to a solely N-or S-oxidizing enzyme by a single R292 mutation (original) (raw)
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Microbial biotechnology, 2012
This work demonstrates that Acinetobacter radioresistens strain S13 during the growth on medium supplemented with long-chain alkanes as the sole energy source expresses almA gene coding for a Baeyer-Villiger monooxygenase (BVMO) involved in alkanes subterminal oxidation. Phylogenetic analysis placed the sequence of this novel BVMO in the same clade of the prodrug activator ethionamide monooxygenase (EtaA) and it bears only a distant relation to the other known class I BVMO proteins. In silico analysis of the 3D model of the S13 BVMO generated by homology modelling also supports the similarities with EtaA by binding ethionamide to the active site. In vitro experiments carried out with the purified enzyme confirm that this novel BVMO is indeed capable of typical Baeyer-Villiger reactions as well as oxidation of the prodrug ethionamide.
Microbial Biotechnology, 2012
This work demonstrates that Acinetobacter radioresistens strain S13 during the growth on medium supplemented with long-chain alkanes as the sole energy source expresses almA gene coding for a Baeyer-Villiger monooxygenase (BVMO) involved in alkanes subterminal oxidation. Phylogenetic analysis placed the sequence of this novel BVMO in the same clade of the prodrug activator ethionamide monooxygenase (EtaA) and it bears only a distant relation to the other known class I BVMO proteins. In silico analysis of the 3D model of the S13 BVMO generated by homology modelling also supports the similarities with EtaA by binding ethionamide to the active site. In vitro experiments carried out with the purified enzyme confirm that this novel BVMO is indeed capable of typical Baeyer-Villiger reactions as well as oxidation of the prodrug ethionamide.
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.
Recent Developments in the Application of Baeyer–Villiger Monooxygenases as Biocatalysts
ChemBioChem, 2010
Baeyer–Villiger monooxygenases (BVMOs) represent a specific class of monooxygenases that are capable of catalyzing a variety of oxidation reactions, including Baeyer–Villiger oxidations. The recently elucidated BVMO crystal structures have provided a more detailed insight into the complex mechanism of these flavin‐containing enzymes. Biocatalytic studies on a number of newly discovered BVMOs have shown that they are very potent oxidative biocatalysts. In addition to catalyzing the regio‐ and enantioselective Baeyer–Villiger oxidations of a wide range of carbonylic compounds, epoxidations, and enantioselective sulfoxidations have also been shown to be part of their catalytic repertoire. This review provides an overview on the recent developments in BVMO‐mediated biocatalytic processes, identification of the catalytic role of these enzymes in metabolic routes and prodrug activation, as well as the efforts in developing effective biocatalytic methodologies to apply BVMOs for the synthe...
Biochemistry, 2016
In the biosynthesis of pentalenolactone (1), PenE and PntE, orthologous proteins from Streptomyces exfoliatus and S. arenae, respectively, catalyze the flavin-dependent Baeyer-Villiger oxidation of 1-deoxy-11-oxopentalenic acid (4) to the lactone pentalenolactone D (5), in which the less-substituted methylene carbon has migrated. By contrast, the paralogous PtlE enzyme from S. avermitilis catalyzes the oxidation of 4 to neopentalenolactone D (6), in which the more substituted methane substitution has undergone migration. We report the design and analysis of 13 single and multiple mutants of PntE mutants to identify the key amino acids that contribute to the regiospecificity of these two classes of Baeyer-Villiger monooxygenases. The L185S mutation in PntE reversed the observed regiospecificity of PntE such that all recombinant PntE mutants harboring this L185S mutation acquired the characteristic regiospecificity of PtlE, catalyzing the conversion of 4 to 6 as the major product. The...
ACS Catalysis, 2022
The typically low thermodynamic and kinetic stability of enzymes is a bottleneck for their application in industrial synthesis. Baeyer–Villiger monooxygenases, which oxidize ketones to lactones using aerial oxygen, among other activities, suffer particularly from these instabilities. Previous efforts in protein engineering have increased thermodynamic stability but at the price of decreased activity. Here, we solved this trade-off by introducing mutations in a cyclohexanone monooxygenase from Acinetobacter sp., guided by a combination of rational and structure-guided consensus approaches. We developed variants with improved activity (1.5- to 2.5-fold) and increased thermodynamic (+5 °C Tm) and kinetic stability (8-fold). Our analysis revealed a crucial position in the cofactor binding domain, responsible for an 11-fold increase in affinity to the flavin cofactor, and explained using MD simulations. This gain in affinity was compatible with other mutations. While our study focused on a particular model enzyme, previous studies indicate that these findings are plausibly applicable to other BVMOs, and possibly to other flavin-dependent monooxygenases. These new design principles can inform the development of industrially robust, flavin-dependent biocatalysts for various oxidations.
Exploring the Structural Basis of Substrate Preferences in Baeyer-Villiger Monooxygenases
Journal of Biological Chemistry, 2012
Background: Bacterial steroid monooxygenase degrades progesterone. Results: The crystallographic and mutagenesis analysis outline a robust active-site scaffold, capable of performing Baeyer-Villiger oxidations on chemically diverse molecules. Conclusion: This and related enzymes represent a fascinating case for the comparative evaluation of user tailored protein engineering with enzyme variants arising through evolution. Significance: These findings highlight the biocatalytic potential of Baeyer-Villiger monooxygenases. 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 proges-terone. 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. * This work was supported by European Union Project Oxygreen number 212281 and Fondazione Cariplo number 2008.3148. □ S This article contains supplemental Figs. S1 and S2. The atomic coordinates and structure factors (codes 4AOX, 4AOS, 4AP1, and 4AP3) have been deposited in the Protein Data Bank,
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.
Antimicrobial agents and chemotherapy, 2015
Antimicrobial resistance is a global issue currently resulting in fatalities of hundreds of thousands a year worldwide. Data present in literature illustrate the emergence of many bacterial species that display resistance to known antibiotics; Acinetobacter spp. is a good example of this. We report here that Acinetobacter radioresistens has a Baeyer-Villiger monooxygenase (Ar-BVMO) with 100% amino acid sequence identity to Ethionamide monooxygenase of the MultiDrug-Resistant (MDR) Acinetobacter baumannii. Both enzymes are phylogenetically only distantly related to other canonical bacterial BVMO proteins. Ar-BVMO is not only capable of oxidizing two anticancer drugs metabolized by human FMO3, Danusertib and Tozasertib, but can also oxidize other synthetic drugs such as Imipenem. The latter is a member of carbapenems, a clinically important antibiotic family used in the treatment of MDR bacterial infections. Susceptibility tests performed by the Kirby-Bauer disk diffusion method demon...
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.