A Method for Screening Baeyer-Villiger Monooxygenase Activity Against Monocyclic Ketones

The assay for Baeyer-Villiger monooxygenase (BVMO) enzyme activity has relied to date on the spectrophotometric change observed on the oxidation of the nicotinamide cofactor during the enzymatic reaction. By analogy to the cyclohexanol catabolic pathway of Acinetobacter calcoaceticus NCIMB 9871, we have developed a specific colorimetric screening method that utilises an esterase to cleave the lactone that is formed in the BVMO reaction. When carried out in a non-buffered or weakly buffered system the resultant change in pH can be visually detected. This allows the rapid assaying and screening of BVMO enzymes. This has been demonstrated with cyclohexanone monooxygenase from A. calcoaceticus. The resultant colour change has been visualised with washed cell suspensions, individual bacterial colonies on Petri dishes and with semi-purified recombinant enzyme utilising Linbro dishes.     

Cloning,expression, and characterization of a Baeyer–Villiger monooxygenase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> DSM 50106 in <Emphasis Type="Italic">E. coli</Emphasis>

A gene encoding a Baeyer–Villiger monooxygenase (BVMO) identified in Pseudomonas fluorescens DSM 50106 was cloned and functionally expressed in Escherichia coli JM109. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis showed an estimated 56 kDa-size protein band corresponding to the recombinant enzyme. Expression in BL21 (DE3) resulted mainly in the formation of inclusion bodies. This could be overcome by coexpression of molecular chaperones, especially the DnaK/DnaJ/GrpE complex, leading to increased production of soluble BVMO enzyme in recombinant E. coli. Examination of the substrate spectra using whole-cell biocatalysis revealed a high specificity of the BVMO for aliphatic open-chain ketones. Thus, octyl acetate, heptyl propionate, and hexyl butyrate were quantitatively formed from the corresponding ketone substrates. Several other esters were obtained in conversion >68%. Selected esters were also produced on preparative scale.     

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).     

Discovery of a thermostable Baeyer–Villiger monooxygenase by genome mining

Baeyer–Villiger monooxygenases represent useful biocatalytic tools, as they can catalyze reactions which are difficult to achieve using chemical means. However, only a limited number of these atypical monooxygenases are available in recombinant form. Using a recently described protein sequence motif, a putative Baeyer–Villiger monooxygenase (BVMO) was identified in the genome of the thermophilic actinomycete Thermobifida fusca. Heterologous expression of the respective protein in Escherichia coli and subsequent enzyme characterization showed that it indeed represents a BVMO. The NADPH-dependent and FAD-containing monooxygenase is active with a wide range of aromatic ketones, while aliphatic substrates are also converted. The best substrate discovered so far is phenylacetone (k_cat = 1.9 s^–1, K_M = 59 M). The enzyme exhibits moderate enantioselectivity with -methylphenylacetone (enantiomeric ratio of 7). In addition to Baeyer–Villiger reactions, the enzyme is able to perform sulfur oxidations. Different from all known BVMOs, this newly identified biocatalyst is relatively thermostable, displaying an activity half-life of 1 day at 52°C. This study demonstrates that, using effective annotation tools, genomes can efficiently be exploited as a source of novel BVMOs.     

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.     

Identification of a novel Baeyer‐Villiger monooxygenase from Acinetobacter radioresistens: close relationship to the Mycobacterium tuberculosis prodrug activator EtaA

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.     

Rapid conversion of cyclohexenone,cyclohexanone and cyclohexanol to ε-caprolactone by whole cells of Geotrichum candidum CCT 1205

?-Caprolactone (?-CL) was obtained with excellent conversion and short reaction times from the substrates cyclohexenone, cyclohexanone and cyclohexanol using whole cells of Brazilian Geotrichum candidum (CCT 1205). The reactions were monitored over time by gas chromatography, and the intermediates of the one-pot cascade biotransformation involving reductions of C=C and C=O bonds as well as the Baeyer–Villiger oxidation were identified and quantified. The Baeyer–Villiger monooxygenase (BVMO) enzyme was predominant, and all three substrates were completely converted into ?-CL. Furthermore, the whole cells of Geotrichum candidum were recycled and reutilized in the biotransformation of cyclohexanone, producing ?-CL at least six consecutive times without a significant loss of activity, reaction yields or product purity.     

A Method for Screening Baeyer-Villiger Monooxygenase Activity Against Monocyclic Ketones

The assay for Baeyer-Villiger monooxygenase (BVMO) enzyme activity has relied to date on the spectrophotometric change observed on the oxidation of the nicotinamide cofactor during the enzymatic reaction. By analogy to the cyclohexanol catabolic pathway of Acinetobacter calcoaceticus NCIMB 9871, we have developed a specific colorimetric screening method that utilises an esterase to cleave the lactone that is formed in the BVMO reaction. When carried out in a non-buffered or weakly buffered system the resultant change in pH can be visually detected. This allows the rapid assaying and screening of BVMO enzymes. This has been demonstrated with cyclohexanone monooxygenase from A. calcoaceticus. The resultant colour change has been visualised with washed cell suspensions, individual bacterial colonies on Petri dishes and with semi-purified recombinant enzyme utilising Linbro dishes.     

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.     

Pseudomonad cyclopentadecanone monooxygenase displaying an uncommon spectrum of Baeyer-Villiger oxidations of cyclic ketones

Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that offer the prospect of high chemo-, regio-, and enantioselectivity in the organic synthesis of lactones or esters from a variety of ketones. In this study, we have cloned, sequenced, and overexpressed in Escherichia coli a new BVMO, cyclopentadecanone monooxygenase (CpdB or CPDMO), originally derived from Pseudomonas sp. strain HI-70. The 601-residue primary structure of CpdB revealed only 29% to 50% sequence identity to those of known BVMOs. A new sequence motif, characterized by a cluster of charged residues, was identified in a subset of BVMO sequences that contain an N-terminal extension of approximately 60 to 147 amino acids. The 64-kDa CPDMO enzyme was purified to apparent homogeneity, providing a specific activity of 3.94 micromol/min/mg protein and a 20% yield. CPDMO is monomeric and NADPH dependent and contains approximately 1 mol flavin adenine dinucleotide per mole of protein. A deletion mutant suggested the importance of the N-terminal 54 amino acids to CPDMO activity. In addition, a Ser261Ala substitution in a Rossmann fold motif resulted in an improved stability and increased affinity of the enzyme towards NADPH compared to the wild-type enzyme (K(m) = 8 microM versus K(m) = 24 microM). Substrate profiling indicated that CPDMO is unusual among known BVMOs in being able to accommodate and oxidize both large and small ring substrates that include C(11) to C(15) ketones, methyl-substituted C(5) and C(6) ketones, and bicyclic ketones, such as decalone and beta-tetralone. CPDMO has the highest affinity (K(m) = 5.8 microM) and the highest catalytic efficiency (k(cat)/K(m) ratio of 7.2 x 10(5) M(-1) s(-1)) toward cyclopentadecanone, hence the Cpd designation. A number of whole-cell biotransformations were carried out, and as a result, CPDMO was found to have an excellent enantioselectivity (E > 200) as well as 99% S-selectivity toward 2-methylcyclohexanone for the production of 7-methyl-2-oxepanone, a potentially valuable chiral building block. Although showing a modest selectivity (E = 5.8), macrolactone formation of 15-hexadecanolide from the kinetic resolution of 2-methylcyclopentadecanone using CPDMO was also demonstrated.     

Isolation and initial characterization of a novel type of Baeyer–Villiger monooxygenase activity from a marine microorganism

A novel type of Baeyer–Villiger monooxygenase (BVMO) has been found in a marine strain of Stenotrophomonas maltophila strain PML168 that was isolated from a temperate intertidal zone. The enzyme is able to use NADH as the source of reducing power necessary to accept the atom of diatomic oxygen not incorporated into the oxyfunctionalized substrate. Growth studies have establish that the enzyme is inducible, appears to serve a catabolic role, and is specifically induced by one or more unidentified components of seawater as well as various anthropogenic xenobiotic compounds. A blast search of the primary sequence of the enzyme, recovered from the genomic sequence of the isolate, has placed this atypical BVMO in the context of the several hundred known members of the flavoprotein monooxygenase superfamily. A particular feature of this BVMO lies in its truncated C‐terminal domain, which results in a relatively small protein (357 amino acids; 38.4 kDa). In addition, metagenomic screening has been conducted on DNA recovered from an extensive range of marine environmental samples to gauge the relative abundance and distribution of similar enzymes within the global marine microbial community. Although low, abundance was detected in samples from many marine provinces, confirming the potential for biodiscovery in marine microorganisms.     

Completing the series of BVMOs involved in camphor metabolism of Pseudomonas putida NCIMB 10007 by identification of the two missing genes, their functional expression in E. coli, and biochemical characterization

The camphor-degrading Baeyer?CVilliger monooxygenases (BVMOs) from Pseudomonas putida NCIMB 10007 have been of interest for over 40?years. So far the FMN- and NADH-dependent type II BVMO 3,6-diketocamphane 1,6-monooxygenase (3,6-DKCMO) and the FAD- and NADPH-dependent type I BVMO 2-oxo-?³-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) have not been entirely studied, since it was not possible to produce those enzymes in satisfactory amounts and purity. In this study, we were able to clone and recombinantly express both enzymes and subsequently use them as biocatalysts for various mono- and bicyclic ketones. Full conversion could be reached with both enzymes towards (±)-cis-bicyclo[3.2.0]hept-2-en-6-one and with 3,6-DKCMO towards (?)-camphor. Further OTEMO gave full conversion with norcamphor. OTEMO was found to have a pH optimum of 9 and a temperature optimum of 20?°C and converted (±)-cis-bicyclo[3.2.0]hept-2-en-6-one with a k _cat/K _M value of 49.3?mM^?1?s^?1.     

Pseudomonad Cyclopentadecanone Monooxygenase Displaying an Uncommon Spectrum of Baeyer-Villiger Oxidations of Cyclic Ketones

Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that offer the prospect of high chemo-, regio-, and enantioselectivity in the organic synthesis of lactones or esters from a variety of ketones. In this study, we have cloned, sequenced, and overexpressed in Escherichia coli a new BVMO, cyclopentadecanone monooxygenase (CpdB or CPDMO), originally derived from Pseudomonas sp. strain HI-70. The 601-residue primary structure of CpdB revealed only 29% to 50% sequence identity to those of known BVMOs. A new sequence motif, characterized by a cluster of charged residues, was identified in a subset of BVMO sequences that contain an N-terminal extension of ~60 to 147 amino acids. The 64-kDa CPDMO enzyme was purified to apparent homogeneity, providing a specific activity of 3.94 μmol/min/mg protein and a 20% yield. CPDMO is monomeric and NADPH dependent and contains ~1 mol flavin adenine dinucleotide per mole of protein. A deletion mutant suggested the importance of the N-terminal 54 amino acids to CPDMO activity. In addition, a Ser261Ala substitution in a Rossmann fold motif resulted in an improved stability and increased affinity of the enzyme towards NADPH compared to the wild-type enzyme (K_m = 8 μM versus K_m = 24 μM). Substrate profiling indicated that CPDMO is unusual among known BVMOs in being able to accommodate and oxidize both large and small ring substrates that include C₁₁ to C₁₅ ketones, methyl-substituted C₅ and C₆ ketones, and bicyclic ketones, such as decalone and β-tetralone. CPDMO has the highest affinity (K_m = 5.8 μM) and the highest catalytic efficiency (k_cat/K_m ratio of 7.2 × 10⁵ M⁻¹ s⁻¹) toward cyclopentadecanone, hence the Cpd designation. A number of whole-cell biotransformations were carried out, and as a result, CPDMO was found to have an excellent enantioselectivity (E > 200) as well as 99% S-selectivity toward 2-methylcyclohexanone for the production of 7-methyl-2-oxepanone, a potentially valuable chiral building block. Although showing a modest selectivity (E = 5.8), macrolactone formation of 15-hexadecanolide from the kinetic resolution of 2-methylcyclopentadecanone using CPDMO was also demonstrated.     

Kinetic mechanism of phenylacetone monooxygenase 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 ( k cat = 3.1 s (-1)), rapid binding of NADPH ( K d = 0.7 microM) is followed by a transfer of the 4( R)-hydride from NADPH to the FAD cofactor ( k red = 12 s (-1)). The reduced PAMO is rapidly oxygenated by molecular oxygen ( k ox = 870 mM (-1) s (-1)), yielding a C4a-peroxyflavin. The peroxyflavin enzyme intermediate reacts with phenylacetone to form benzylacetate ( k 1 = 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. This study shows that kinetic behavior of PAMO is significantly different when compared with that of sequence-related monooxygenases, e.g., cyclohexanone monooxygenase and liver microsomal flavin-containing monooxygenase. Inspection of the crystal structure of PAMO has revealed that residue R337, which is conserved in other BVMOs, is positioned close to the flavin cofactor. The analyzed R337A and R337K mutant enzymes were still able to form and stabilize the C4a-peroxyflavin intermediate. The mutants were unable to convert either phenylacetone or benzyl methyl sulfide. This demonstrates that R337 is crucially involved in assisting PAMO-mediated Baeyer-Villiger and sulfoxidation reactions.     

Nitration of PPARgamma inhibits ligand-dependent translocation into the nucleus in a macrophage-like cell line,RAW 264

Baeyer-Villiger monooxygenases (BVMOs) form a distinct class of flavoproteins that catalyze the insertion of an oxygen atom in a C-C bond using dioxygen and NAD(P)H. Using newly characterized BVMO sequences, we have uncovered a BVMO-identifying sequence motif: FXGXXXHXXXW(P/D). Studies with site-directed mutants of 4-hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB suggest that this fingerprint sequence is critically involved in catalysis. Further sequence analysis showed that the BVMOs belong to a novel superfamily that comprises three known classes of FAD-dependent monooxygenases: the so-called flavin-containing monooxygenases (FMOs), the N-hydroxylating monooxygenases (NMOs), and the BVMOs. Interestingly, FMOs contain an almost identical sequence motif when compared to the BVMO sequences: FXGXXXHXXX(Y/F). Using these novel amino acid sequence fingerprints, BVMOs and FMOs can be readily identified in the protein sequence databank.     

mRNA Differential Display in a Microbial Enrichment Culture: Simultaneous Identification of Three Cyclohexanone Monooxygenases from Three Species

mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli.     

Studies on fungi in the root region

Summary Fungal isolations were made from roots ofPhaseolus vulgaris after washing in sterile water, at monthly intervals throughout the life of the plant, and from other roots after dissection and after surface sterilization at certain plant ages only. A table is provided showing the relative importance of the most common species isolated in each of four clearly distinct microhabitats — the root surface, the cortex, the outer stele and the inner stele.Fusarium oxysporum andCylindrocarpon radicicola were the most frequently isolated fungi from the roots.Fusarium oxysporum was most abundant on young roots and seemed to be associated, particularly, with the root surface and cortical tissues.Cylindrocarpon radicicola, although common on young roots, was more abundant on older roots and was an important initial colonist of the stelar tissues. Sterile mycelia were isolated mainly from older roots and seemed to be responsible, withC. radicicola, for the initial colonization of the stele. Microscopic examination of roots showed the cortical tissues to be increasingly penetrated by fungal hyphae with plant age but extensive fungal penetration of the endodermis and stelar tissues did not occur until the plants were at least five months old.     

Biocatalyst assessment of recombinant whole-cells expressing the Baeyer-Villiger monooxygenase from Xanthobacter sp. ZL5

An Escherichia coli-based expression system for the Baeyer-Villiger monooxygenase (BVMO) from Xanthobacter sp. ZL5 was screened for whole-cell-mediated biotransformations. Biooxidation studies included kinetic resolutions and regiodivergent conversions of structurally diverse cycloketones. An extended phylogenetic analysis of the BVMOs currently available as recombinant systems with experimentally determined Baeyer-Villigerase activity showed that the enzyme originating from Xanthobacter sp. ZL5 clusters together with the sequences of bacterial CHMO-type BVMOs. The regio- and enantiopreferences experimentally observed for this enzyme are clearly similar to the biocatalytic performance of cyclohexanone monooxygenase from Acinetobacter as prototype for this group of BVMOs and support our previously reported family grouping.     

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.