Substrate Specificity and Enantioselectivity of 4-Hydroxyacetophenone Monooxygenase

The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained [77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee >99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications.     

4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB. A novel flavoprotein catalyzing Baeyer-Villiger oxidation of aromatic compounds.

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 approximately 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 approximately 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.     

Continuous testing system for Baeyer-Villiger biooxidation using recombinant Escherichia coli expressing cyclohexanone monooxygenase encapsulated in polyelectrolyte complex capsules

An original strategy for universal laboratory testing of Baeyer-Villiger monooxygenases based on continuous packed-bed minireactor connected with flow calorimeter and integrated with bubble-free oxygenation is reported. Model enantioselective Baeyer-Villiger biooxidations of rac-bicyclo[3.2.0]hept-2-en-6-one to corresponding lactones (1R,5S)-3-oxabicyclo-[3.3.0]oct-6-en-3-one and (1S,5R)-2-oxabicyclo-[3.3.0]oct-6-en-3-one as important chiral synthons for the synthesis of bioactive compounds were performed in the minireactor equipped with a column packed with encapsulated recombinant cells Escherichia coli overexpressing cyclohexanone monooxygenase. The cells were encapsulated in polyelectrolyte complex capsules formed by reaction of oppositely charged polymers utilizing highly reproducible and controlled encapsulation process. Encapsulated cells tested in minireactor exhibited high operational stability with 4 complete substrate conversions to products and 6 conversions above 80% within 14 repeated consecutive biooxidation tests. Moreover, encapsulated cells showed high enzyme stability during 91 days of storage with substrate conversions above 80% up to 60 days of storage. Furthermore, usable thermometric signal of Baeyer-Villiger biooxidation obtained by flow calorimetry using encapsulated cells was utilized for preparatory kinetic study in order to guarantee sub-inhibitory initial substrate concentration for biooxidation tests.     

Coenzyme binding during catalysis is beneficial for the stability of 4-hydroxyacetophenone monooxygenase

The NADPH-dependent dimeric flavoenzyme 4-hydroxyacetophenone monooxygenase (HAPMO) catalyzes Baeyer-Villiger oxidations of a wide range of ketones, thereby generating esters or lactones. In the current work, we probed HAPMO-coenzyme complexes present during the enzyme catalytic cycle with the aim to gain mechanistic insight. Moreover, we investigated the structural role of the nicotinamide coenzyme. For these studies, we used (i) wild type HAPMO, (ii) the R339A variant, which is active but has a low affinity toward NADPH, and (iii) the R440A variant, which is inactive but has a high affinity toward NADPH. Electrospray ionization mass spectrometry was used as the primary tool to directly observe noncovalent protein-coenzyme complexes in real time. These analyzes showed for the first time that the nicotinamide coenzyme remains bound to HAPMO during the entire catalytic cycle of the NADPH oxidase reaction. This may also have implications for other homologous Baeyer-Villiger monooxygenases. Together with the observations that NADP(+) only weakly interacts with oxidized enzyme and that HAPMO is mainly in the reduced form during catalysis, we concluded that NADP(+) interacts tightly with the reduced form of HAPMO. We also demonstrated that the association with the coenzyme is crucial for enzyme stability. The interaction with the coenzyme analog 3-aminopyridine adenine dinucleotide phosphate (AADP(+)) strongly enhanced the thermal stability of wild type HAPMO. This coenzyme-induced stabilization may also be important for related enzymes.     

Identifying determinants of NADPH specificity in Baeyer-Villiger monooxygenases.

The Baeyer-Villiger monooxygenase (BVMO), 4-hydroxyacetophenone monooxygenase (HAPMO), uses NADPH and O(2) to oxidize a variety of aromatic ketones and sulfides. The FAD-containing enzyme has a 700-fold preference for NADPH over NADH. Sequence alignment with other BVMOs, which are all known to be selective for NADPH, revealed three conserved basic residues, which could account for the observed coenzyme specificity. The corresponding residues in HAPMO (Arg339, Lys439 and Arg440) were mutated and the properties of the purified mutant enzymes were studied. For Arg440 no involvement in coenzyme recognition could be shown as mutant R440A was totally inactive. Although this mutant could still be fully reduced by NADPH, no oxygenation occurred, indicating that this residue is crucial for completing the catalytic cycle of HAPMO. Characterization of several Arg339 and Lys439 mutants revealed that these residues are indeed both involved in coenzyme recognition. Mutant R339A showed a largely decreased affinity for NADPH, as judged from kinetic analysis and binding experiments. Replacing Arg339 also resulted in a decreased catalytic efficiency with NADH. Mutant K439A displayed a 100-fold decrease in catalytic efficiency with NADPH, mainly caused by an increased K(m). However, the efficiency with NADH increased fourfold. Saturation mutagenesis at position 439 showed that the presence of an asparagine or a phenylalanine improves the catalytic efficiency with NADH by a factor of 6 to 7. All Lys439 mutants displayed a lower affinity for AADP(+), confirming a role of the lysine in recognizing the 2'-phosphate of NADPH. The results obtained could be extrapolated to the sequence-related cyclohexanone monooxygenase. Replacing Lys326 in this BVMO, which is analogous to Lys439 in HAPMO, again changed the coenzyme specificity towards NADH. These results indicate that the strict NADPH dependency of this class of monooxygenases is based upon recognition of the coenzyme by several basic residues.     

Microbial transformations 59: first kilogram scale asymmetric microbial Baeyer-Villiger oxidation with optimized productivity using a resin-based in situ SFPR strategy

This study is demonstrating the scale up of asymmetric microbial Baeyer-Villiger oxidation of racemic bicyclo[3.2.0]hept-2-en-6-one (1) to the kilogram scale using a 50 L bioreactor. The process has been optimized with respect to bottlenecks identified in downscaled experiments. A high productivity was obtained combining a resin-based in situ substrate feeding and product removal methodology (in situ SFPR), a glycerol feed control, and an improved oxygenation device (using a sintered-metal sparger). As expected both regioisomeric lactones [(-)-(1S,5R)-2 and (-)-(1R,5S)-3] were obtained in nearly enantiopure form (ee > 98%) and good yield. This represents the first example of such an asymmetric Baeyer-Villiger biooxidation reaction ever operated at that scale. This novel resin-based in situ SFPR technology therefore clearly opens the way to further (industrial) upscaling of this highly valuable (asymmetric) reaction.     

Conversion of 4-hydroxyacetophenone into 4-phenyl acetate by a flavin adenine dinucleotide-containing Baeyer-Villiger-type monooxygenase

An arylketone monooxygenase was purified from Pseudomonas putida JD1 by ion exchange and affinity chromatography. It had the characteristics of a Baeyer-Villiger-type monooxygenase and converted its substrate, 4-hydroxyacetophenone, into 4-hydroxyphenyl acetate with the consumption of one molecule of oxygen and oxidation of one molecule of NADPH per molecule of substrate. The enzyme was a monomer with an M(r) of about 70,000 and contained one molecule of flavin adenine dinucleotide (FAD). The enzyme was specific for NADPH as the electron donor, and spectral studies showed rapid reduction of the FAD by NADPH but not by NADH. Other arylketones were substrates, including acetophenone and 4-hydroxypropiophenone, which were converted into phenyl acetate and 4-hydroxyphenyl propionate, respectively. The enzyme displayed Michaelis-Menten kinetics with apparent K(m) values of 47 microM for 4-hydroxyacetophenone, 384 microM for acetophenone, and 23 microM for 4-hydroxypropiophenone. The apparent K(m) value for NADPH with 4-hydroxyacetophenone as substrate was 17.5 microM. The N-terminal sequence did not show any similarity to other proteins, but an internal sequence was very similar to part of the proposed NADPH binding site in the Baeyer-Villiger monooxygenase cyclohexanone monooxygenase from an Acinetobacter sp.     

Use of isolated cyclohexanone monooxygenase from recombinant Escherichia coli as a biocatalyst for Baeyer-Villiger and sulfide oxidations

The performance, in Baeyer-Villiger and heteroatom oxidations, of a partially purified preparation of cyclohexanone monooxygenase obtained from an Escherichia coli strain in which the gene of the enzyme was cloned and overexpressed was investigated. As model reactions, the oxidations of racemic bicyclo[3.2.0]hept-2-en-6-one into two regioisomeric lactones and of methyl phenyl sulphide into the corresponding (R)-sulphoxide were used. Enzyme stability and reuse, substrate and product inhibition, product removal, and cofactor recycling were evaluated. Of the various NADPH regeneration systems tested, 2-propanol/alcohol dehydrogenase from Thermoanerobium brockii appeared the most suitable because of the low cost of the second substrate and the high regeneration rate. Concerning enzyme stability, kosmotropic salts were the only additives able to improve it (e.g., half-life from 1 day in diluted buffer to 1 week in 1 M sodium sulphate) but only under storage conditions. Instead, significant stabilization under working conditions was obtained by immobilization on Eupergit C (half-life approximately 2.5 days), a procedure that made it possible to reuse the catalyst up to 16 times with complete substrate (5 g x L(-1)) conversion at each cycle. Reuse of free enzyme was also achieved in a membrane reactor but with lower efficiency. Water-organic solvent biphasic systems, which would overcome substrate inhibition and remove from the aqueous phase, where reaction takes place, the formed product, were unsuccessful because of their destabilizing effect on cyclohexanone monooxygenase. More satisfactory was continuous substrate feeding, which shortened reaction times and, very importantly, yielded in the case of bicyclo[3.2.0]hept-2-en-6-one (10 g x L(-1)) both lactone products with high optical purity (enantiomeric excess > or = 96%), which was not the case when all of the substrate was added in a single batch.     

Reactor operation and scale-up of whole cell Baeyer-Villiger catalyzed lactone synthesis

The recombinant whole cell biocatalyst Escherichia coli TOP10 [pQR239], expressing cyclohexanone monooxygenase from Acinetobacter calcoaceticus NCIMB 9871, was used in 1.5- and 55-L fed-batch processes to oxidize bicyclo[3.2.0]hept-2-en-6-one to its corresponding regioisomeric lactones, (-)-(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and (-)-(1R,5S)-3-oxabicyclo[3.3.0]oct-6-en-2-one. By employing a bicyclo[3.2.0]hept-2-en-6-one feed rate below that of the theoretical volumetric biocatalyst activity (275 micromol x min(-1) x L(-1)), the reactant concentration in the bioreactor was successfully maintained below the inhibitory concentration of 0.2-0.4 g x L(-1). In this way approximately 3.5 g x L(-1) of the combined regioisomeric lactones was produced with a yield of product on reactant of 85-90%. The key limitation to the process was shown to be product inhibition. This process was scaled up to 55 L, producing over 200 g of combined lactone product. Using a simple downstream process (centrifugation, adsorption to activated charcoal, 5-fold concentration with ethyl acetate elution, and silica gel chromatography), we have shown that the two regioisomeric lactone products could be isolated and purified at this scale.     

Discovery of a novel styrene monooxygenase originating from the metagenome

Oxygenases form an interesting class of biocatalysts, as they typically perform oxygenations with exquisite chemo-, regio-, and/or enantioselectivity. It has been observed that, once heterologously expressed in Escherichia coli, some oxygenases are able to form the blue pigment indigo. We have exploited this characteristic to screen a metagenomic library derived from loam soil and identified a novel oxygenase. This oxygenase shows 50% sequence identity with styrene monooxygenases from pseudomonads (StyA). Only a limited number of homologs can be found in the genome sequence database, indicating that this biocatalyst is a member of a relatively small family of bacterial monooxygenases. The newly identified monooxygenase catalyzes the epoxidation of styrene and styrene derivatives and forms the corresponding (S)-epoxides with excellent enantiomeric excess [e.g., (S)-styrene oxide is formed with >99% enantiomeric excess, ee] and therefore is named styrene monooxgenase subunit A (SmoA). SmoA shows high enantioselectivity towards aromatic sulfides [e.g., (R)-ethyl phenyl sulfoxide is formed with 92% ee]. This excellent enantioselectivity in combination with the moderate sequence identity forms a clear indication that SmoA from a metagenomic origin represents a new enzyme within the small family of styrene monooxygenases.     

Biotransformations by microbial Baeyer-Villiger monooxygenases stereoselective lactone formationin vitro by coupled enzyme systems

Summary The regio- and stereoselective biotransformation of bicyclo (3. 2. 0) hept-2-en-6-one by the NADH-dependent Baeyer-Villiger monooxygenase from camphorgrownPseudomonas putida NCIMB 10007 has been shown to yield a chiral lactone not accessible by curently-used biocatalysts. The biotransformation can be conductedin vitro using two alternative coupled enzyme systems (alcohol dehydrogenase and monooxygenase: formate dehydrogenase and monooxygenase) within situ recycling of NAD⁺/NADH.     

Novel side chain modified Delta8(14)-15-ketosterols

Synthesis of five novel Delta8(14)-15-ketosterols comprising modified side chains starting from ergosterol is described. Ergosteryl acetate was converted into (22E)-3beta-acetoxy-5alpha-ergosta-8(14),22-dien-15-one through three stages in 32% overall yield; further transformations of the product obtained led to (22E)-3beta-hydroxy-5alpha-ergosta-8(14),22-dien-15-one, (22S,23S)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one, (22R,23R)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one, (22R,23R)-5alpha-ergost-8(14)-en-15-on-3beta,22,23-triol and (22R,23R)-3beta-hydroxy-22,23-isopropylidenedioxy-5alpha-ergost-8(14)-en-15-one. New Delta8(14)-15-ketosterols were evaluated for their cytotoxicity and effects on sterol biosynthesis in human hepatoma Hep G2 cells in comparison with known 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. Among the compounds tested, (22R,23R)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one was found to be the most potent inhibitor of sterol biosynthesis (IC(50)=0.6+/-0.2microM), whereas (22R,23R)-5alpha-ergost-8(14)-en-15-on-3beta,22,23-triol exhibited the highest cytotoxicity (TC(50)=12+/-3microM at a 24h incubation).     

The analysis and application of a recombinant monooxygenase library as a biocatalyst for the Baeyer-Villiger reaction

Because of their selectivity and catalytic efficiency, BVMOs are highly valuable biocatalysts for the chemoenzymatic synthesis of a broad range of useful compounds. In this study, we investigated the microbial Baeyer-Villiger oxidation and sulfoxidation of thioanisole and bicyclo[3.2.0]hept-2-en-6-one using whole Escherichia coli cells that recombined with each of the Baeyer-Villiger monooxygenases originated from Pseudomonas aeruginosa PAO1 and two from Streptomyces coelicolor A3(2). The three BVMOs were identified in the microbial genome database by a recently described protein sequence motif; e.g., BVMO motif (FXGXXXHXXXW). The reaction products were identified as (R)-/(S)sulfoxide and 2-oxabicyclo/3-oxabicyclo[3.3.0]oct-6-en-2-one by GC-MS analysis. Consequently, this study demonstrated that the three enzymes can indeed catalyze the Baeyer-Villiger reaction as a biocatalyst, and effective annotation tools can be efficiently exploited as a source of novel BVMOs.     

Oxidations by microbial NADH plus FMN-dependent luciferases from Photobacterium phosphoreum and Vibrio fischeri

The NADH plus FMN-dependent luciferase from Photobacterium phosphoreum NCIMB 844 has been shown to act as a Baeyer-Villiger monooxygenase able to perform regio-, and where relevant, enantioselective biotransformations of various xenobiotic aliphatic and alicyclic ketones by nucleophilic oxygenation. The useful lactone (−)-(1S,5R)-2-oxabicyclo [3.3.0]oct-6-en-3-one was produced with high optical purity (> 95% ee). A similar biotransformation was recorded with the equivalent luciferase from Vibrio fischeri ATCC 7744.     

Preparative scale Baeyer-Villiger biooxidation at high concentration using recombinant Escherichia coli and in situ substrate feeding and product removal process

An efficient biocatalytic process based on the use of adsorbent resin (in situ substrate feeding and product removal) makes experiments at high substrate concentration possible by overcoming limitations due to substrate and product inhibition. This process was successfully applied to the preparative scale Baeyer-Villiger biooxidation of (-)-(1S,5R)-bicyclo[3.2.0]hept-2-en-6-one (25 g). Whole cells of recombinant E. coli (1 liter) overexpressing cyclohexanone monooxygenase were used as a biocatalyst and the substrate was preloaded onto the adsorbent resin. The corresponding lactone was obtained in 75-80% yield. Time for cell growth and biotransformation is about 24 h each and oxygen supply can be improved by using a tailor-made bubble column.     

The prodrug activator EtaA from Mycobacterium tuberculosis is a Baeyer-Villiger monooxygenase

EtaA is a newly identified FAD-containing monooxygenase that is responsible for activation of several thioamide prodrugs in Mycobacterium tuberculosis. It was found that purified EtaA displays a remarkably low activity with the antitubercular prodrug ethionamide. Hinted by the presence of a Baeyer-Villiger monooxygenase sequence motif in the EtaA sequence, we have been able to identify a large number of novel EtaA substrates. It was discovered that the enzyme converts a wide range of ketones to the corresponding esters or lactones via a Baeyer-Villiger reaction, indicating that EtaA represents a Baeyer-Villiger monooxygenase. With the exception of aromatic ketones (phenylacetone and benzylacetone), long-chain ketones (e.g. 2-hexanone and 2-dodecanone) also are converted. EtaA is also able to catalyze enantioselective sulfoxidation of methyl-p-tolylsulfide. Conversion of all of the identified substrates is relatively slow with typical k(cat) values of around 0.02 s(-1). The best substrate identified so far is phenylacetone (K(m) = 61 microM, k(cat) = 0.017 s(-1)). Redox monitoring of the flavin cofactor during turnover of phenylacetone indicates that a step in the reductive half-reaction is limiting the rate of catalysis. Intriguingly, EtaA activity could be increased by one order of magnitude by adding bovine serum albumin. This reactivity and substrate acceptance-profiling study provides valuable information concerning this newly identified prodrug activator from M. tuberculosis.     

Chemical synthesis, spectral properties, and chromatography of 4alpha-methyl and 4beta-methyl isomers of (24R)-24-ethyl-5alpha-cholestan-3beta-ol and (24S)-24-ethyl-cholesta-5,22-dien-3beta-ol.

Described herein are chemical syntheses of the following compounds: 4-methyl-(24S)-24-ethyl-cholesta-4,22-dien-3-one, 4,4-dimethyl-(24S)-24-ethyl-cholesta-5,22-dien-3-one, 4beta-methyl-(24R)-24-ethyl-5alpha-cholestan-3beta-ol, 4alpha-methyl-(24R)-24-ethyl-5alpha-cholestan-3beta-ol, 4alpha-methyl-(24S)-24-ethyl-5alpha-cholest-22-en-3beta-ol, 4-methyl-6beta-bromo-(24S)-24-ethyl-cholesta-4,22-dien-3-one, 4alpha-methyl-(24S)-24-ethyl-cholesta-5,22-dien-3beta-ol, 4alpha,5alpha-epoxy-(24S)-24-ethyl-cholesta-4,22-dien-3beta-yl acetate, 4beta-methyl-(24S)-24-ethyl-cholest-22-en-3beta,5alpha-diol, 4beta-methyl-5alpha-hydroxy-(24S)-24-ethyl-cholest-22-en-3beta-yl acetate, 4beta-methyl-(24S)-24-ethyl-cholesta-5,22-dien-3beta-yl acetate and 4beta-methyl-(24S)-24-ethyl-cholesta-5,22-dien-3beta-ol. Chromatographic, nuclear magnetic resonance, and mass spectral data are presented for the compounds under consideration.     

Cloning,Expression, Characterization,and Biocatalytic Investigation of the 4-Hydroxyacetophenone Monooxygenase from Pseudomonas putida JD1

While the number of available recombinant Baeyer-Villiger monooxygenases (BVMOs) has grown significantly over the last few years, there is still the demand for other BVMOs to expand the biocatalytic diversity. Most BVMOs that have been described are dedicated to convert efficiently cyclohexanone and related cyclic aliphatic ketones. To cover a broader range of substrate types and enantio- and/or regioselectivities, new BVMOs have to be discovered. The gene encoding a BVMO identified in Pseudomonas putida JD1 converting aromatic ketones (HAPMO; 4-hydroxyacetophenone monooxygenase) was amplified from genomic DNA using SiteFinding-PCR, cloned, and functionally expressed in Escherichia coli. Furthermore, four other open reading frames could be identified clustered around this HAPMO. It has been suggested that these proteins, including the HAPMO, might be involved in the degradation of 4-hydroxyacetophenone. Substrate specificity studies revealed that a large variety of other arylaliphatic ketones are also converted via Baeyer-Villiger oxidation into the corresponding esters, with preferences for para-substitutions at the aromatic ring. In addition, oxidation of aldehydes and some heteroaromatic compounds was observed. Cycloketones and open-chain ketones were not or poorly accepted, respectively. It was also found that this enzyme oxidizes aromatic ketones such as 3-phenyl-2-butanone with excellent enantioselectivity (E ≫100).Baeyer-Villiger monooxygenases (BVMOs; EC 1.14.13.x) belong to the class of oxidoreductases and convert aliphatic, cyclic, and/or aromatic ketones to esters or lactones, respectively, using molecular oxygen (29). Thus, they mimic the chemical Baeyer-Villiger oxidation, which is usually peracid catalyzed and was first described by Adolf Baeyer and Viktor Villiger in 1899 (2). All characterized BVMOs thus far are NAD(P)H dependent and require flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as prosthetic group, which is crucial for catalysis.Today, BVMOs are increasingly recognized as valuable catalysts for stereospecific oxidation reactions. These enzymes display a remarkably broad acceptance profile for nonnatural substrates. Besides conversion of a wide range of aliphatic open-chain, cyclic, and aromatic ketones, they are also able to oxygenate sulfides (16), selenides (27), amines (33), phosphines, olefins (5), aldehydes, and borone- and iodide-containing compounds (Fig. ​(Fig.1)1) (7).Open in a separate windowFIG. 1.Range of Baeyer-Villiger oxidations catalyzed by BVMOs.Therefore, recombinantly available BVMOs are powerful tools in organic chemistry and demonstrate a high potential as alternatives to existing chemical technologies, where some of these reactions are difficult to perform selectively using chemical catalysts.Except for this promiscuity in reactivity, high enantioselectivities, as well as regio- and stereoselectivities, make them interesting for the pharmaceutical, food, and cosmetic industries, where enantiomerically pure compounds are valuable building blocks. In addition, renunciation of peracids when applying enzymatic driven Baeyer-Villiger oxidations turns them into an ecofriendly alternative and led to a considerable interest for biotransformations using BVMOs on an industrial scale (1, 8, 13-15) during the past decades.Already in 1948 it was recognized that enzymes catalyzing the Baeyer-Villiger reaction exist in nature (39). This was concluded from the observation that a biological Baeyer-Villiger reaction occurred during the degradation of steroids by fungi. Still it took 20 years for the first BVMO to be isolated and characterized (10). Thus far, 22 BVMOs have been cloned, functionally expressed, and characterized. In Fig. ​Fig.22 their genetic relationships are illustrated, and all BVMOs are sorted into different classes on the basis of their substrate specificity. Only two BVMOs, the 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB (19) and phenylacetone monooxygenase (PAMO) from Thermobifida fusca (11), converting arylaliphatic and aromatic ketones were described. The latter is the only thermostable BVMO and served as a model to elucidate the enzymatic mechanism (28).Open in a separate windowFIG. 2.Phylogenetic relationships within BVMOs. The sequences of 22 enzymes with confirmed BVMO activity were aligned, and an unrooted phylogenetic tree was generated using CLUSTAL W (v.1.81). Cycloketone-converting BVMO (solid lines), open-chain ketone-converting BVMO (dashed lines), and arylketone-converting BVMO (dash/dot lines). NCBI accession numbers of protein sequences: CHMO Acinetobacter, CHMO Acinetobacter calcoaceticus NCIMB 9871 (BAA86293); CHMO Xanthobacter, BVMO Xanthobacter sp. strain ZL5 (CAD10801); CHMO Brachymonas, CHMO Brachymonas petroleovorans (AAR99068); CHMO1 Arthrobacter, CHMO1 Arthrobacter sp. strain BP2 (AAN37479); CHMO2 Arthrobacter, CHMO2 Arthrobacter sp. strain L661 (ABQ10653); CHMO1 Rhodococcus, CHMO1 Rhodococcus Phi1 (AAN37494); CHMO2 Rhodococcus, CHMO2 Rhodococcus Phi2 (AAN37491); CHMO1 Brevibacterium, CHMO1 Brevibacterium sp. strain HCU (AAG01289); CHMO2 Brevibacterium, CHMO2 Brevibacterium sp. strain HCU (AAG01290); CPMO Comamonas, cyclopentanone monooxygenase Comamonas sp. strain NCIMB 9872 (BAC22652); CPDMO Pseudomonas, cyclopentadecanone monooxygenase Pseudomonas sp. strain HI-70 (BAE93346); CDMO R. ruber, cyclododecane monooxygenase Rhodococcus ruber SCI (AAL14233); BVMO Mycobacterium tuberculosis Rv3083, BVMO M. tuberculosis H37Rv (gene Rv3083) (CAA16141); BVMO M. tuberculosis Rv3049c, BVMO M. tuberculosis H37Rv (gene Rv3049c) (CAA16134); BVMO M. tuberculosis Rv3854c, BVMO M. tuberculosis H37Rv (gene Rv3854c) (CAB06212); BVMO P. putida KT2440, BVMO P. putida KT2440 (AAN68413); BVMO P. fluorescens DSM50106: BVMO P. fluorescens DSM50106 (AAC36351); BVMO Pseudomonas veronii MEK700, BVMO P. veronii MEK700 (ABI15711); STMO Rhodococcus rhodochrous, steroid monooxygenase R. rhodochrous (BAA24454); PAMO T. fusca, phenylacetone monooxygenase T. fusca (Q47PU3); HAPMO P. fluorescens ACB, 4-hydroxyacetophenone monooxygenase from P. fluorescens ACB (AAK54073); HAPMO P. putida JD1, 4-hydroxyacetophenone monooxygenase from P. putida JD1 (FJ010625 [the present study]).We report here the amplification, cloning, functional expression, and characterization of a HAPMO from Pseudomonas putida JD1 oxidizing a broad range of aromatic ketones and further substrates.     

Three oxygenated cyclohexenone derivatives produced by an endophytic fungus

Three cyclohexenone derivatives, (4S,5S,6S)-5,6-epoxy-4-hydroxy-3-methoxy-5-methyl-cyclohex-2-en-1-one (1), (4R,5R)-4,5-dihydroxy-3-methoxy-5-methyl-cyclohex-2-en-1-one (2), and (4R,5S,6R)-4,5,6-trihydroxy-3-methoxy-5-methyl-cyclohex-2-en-1-one (3), were isolated from unpolished rice fermented with an xylariaceous endophytic fungus (strain YUA-026). The structures of three compounds were established on the basis of spectroscopic analyses and chemical conversion. The minimum inhibitory concentrations of 1 and 3 were 100 microg/ml and 400 microg/ml against Staphylococcus aureus, 100 microg/ml and 200 microg/ml against Pseudomonas aeruginosa, and 200 microg/ml and >400 microg/ml against Candida albicans, respectively. In addition, 1 and 3 exhibited phytotoxic activity against lettuce.     

Baeyer-Villiger monooxygenases in aroma compound synthesis

Baeyer-Villiger monooxygenases (BVMOs) are presented as highly selective and efficient biocatalysts for the synthesis of aroma lactones via kinetic resolution of 2-substituted cycloketones, exemplified with two δ-valerolactones, the jasmine lactones and their ε-caprolactone homologs. Analytical scale screens of our BVMO library ensued by preparative whole-cell biotransformations led to the identification of two enzymes (cyclohexanone monooxygenase from Arthrobacter BP2 and cyclododecanone monooxygenase from Rhodococcus SC1) perfectly suited for the task at hand: easily accessible racemic starting materials were bio-oxidized to almost enantiopure ketones and lactones in good yields (48-74%) and optical purities (ee 93% to >99%, E>100).