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.     

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 eukaryotic cycloalkanone monooxygenase from <Emphasis Type="Italic">Cylindrocarpon radicicola</Emphasis> ATCC 11011

In this study, we have cloned and characterized a cycloalkanone monooxygenase (CAMO) from the ascomycete Cylindrocarpon radicicola ATCC 11011 (identical to Cylindrocarpon destructans DSM 837). The primary structure of this Baeyer–Villiger monooxygenase (BMVO) revealed 531 residues with around 45% sequence identity to known cyclohexanone monooxygenases. The enzyme was functionally overexpressed in Escherichia coli and investigated with respect to substrate spectrum and kinetic parameters. Substrate specificity studies revealed that a large variety of cycloaliphatic and bicycloaliphatic ketones are converted by this CAMO. A high catalytic efficiency against cyclobutanone was observed and seems to be a particular property of this BVMO. The thus produced butyrolactone derivatives are valuable building blocks for the synthesis of a variety of natural products and bioactive compounds. Furthermore, the enzyme revealed activity against open-chain ketones such as cyclobutyl, cyclopentyl and cyclohexyl methyl ketone which have not been reported to be accepted by typical cyclohexanone monooxygenases. These results suggest that the BVMO from C. radicicola indeed might be rather unique and since no BVMOs originating from eukaryotic organisms have been produced recombinantly so far, this study provides the first example for such an enzyme.     

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.     

Cloning of Baeyer-Villiger monooxygenases from Comamonas,Xanthobacter and Rhodococcus using polymerase chain reaction with highly degenerate primers

To clone novel type 1 Baeyer-Villiger monooxygenase (BVMO) genes, we isolated or collected 25 bacterial strains able to grow on alicyclic compounds. Twelve of the bacterial strains yielded polymerase chain reaction (PCR) fragments with highly degenerate primers based on the sequences of known and putative BVMOs. All these fragments were found to encode peptides homologous to published BVMO sequences. The complete BVMO genes and flanking DNA were cloned from a Comamonas, a Xanthobacter and a Rhodococcus strain using the PCR fragments as probes. BVMO genes cloned from the first two strains could be expressed to high levels in Escherichia coli using standard expression vectors, and the recombinants converted cyclopentanone and cyclohexanone to the corresponding lactones. The Rhodococcus BVMO, a putative steroid monooxygenase, could be expressed after modification of the N-terminal sequence. However, recombinants expressing this protein did not show activity towards progesterone. An esterase homologue located directly upstream of the Xanthobacter BVMO gene and a dehydrogenase homologue encoded directly downstream of the Comamonas sp. NCIMB 9872 BVMO gene were also expressed in E. coli and shown to specify lactone hydrolase and cyclohexanol dehydrogenase activity respectively.     

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.     

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.     

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.     

Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT.

4-Methyl-5-nitrocatechol (MNC) is an intermediate in the degradation of 2,4-dinitrotoluene by Burkholderia sp. strain DNT. In the presence of NADPH and oxygen, MNC monooxygenase catalyzes the removal of the nitro group from MNC to form 2-hydroxy-5-methylquinone. The gene (dntB) encoding MNC monooxygenase has been previously cloned and characterized. In order to examine the properties of MNC monooxygenase and to compare it with other enzymes, we sequenced the gene encoding the MNC monooxygenase and purified the enzyme from strain DNT. dntB was localized within a 2.2-kb ApaI DNA fragment. Sequence analysis of this fragment revealed an open reading frame of 1,644 bp with an N-terminal amino acid sequence identical to that of purified MNC monooxygenase from strain DNT. Comparison of the derived amino acid sequences with those of other genes showed that DntB contains the highly conserved ADP and flavin adenine dinucleotide (FAD) binding motifs characteristic of flavoprotein hydroxylases. MNC monooxygenase was purified to homogeneity from strain DNT by anion exchange and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of 60,200, which is consistent with the size determined from the gene sequence. The native molecular weight determined by gel filtration was 65,000, which indicates that the native enzyme is a monomer. It used either NADH or NADPH as electron donors, and NADPH was the preferred cofactor. The purified enzyme contained 1 mol of FAD per mol of protein, which is also consistent with the detection of an FAD binding motif in the amino acid sequence of DntB. MNC monooxygenase has a narrow substrate specificity. MNC and 4-nitrocatechol are good substrates whereas 3-methyl-4-nitrophenol, 3-methyl-4-nitrocatechol, 4-nitrophenol, 3-nitrophenol, and 4-chlorocatechol were not. These studies suggest that MNC monooxygenase is a flavoprotein that shares some properties with previously studied nitrophenol oxygenases.     

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.     

Assessment of the Microbiological Potential for the Natural Attenuation of Petroleum Hydrocarbons in a Shallow Aquifer System

Abstract A multidisciplinary field study investigating the fate and transport of petroleum hydrocarbons commonly associated with jet-fuel contamination is currently underway at Columbus Air Force Base (AFB), Mississippi. Sixty sediment cores from 12 boreholes were recovered from the study aquifer. The goal of this initial sampling was to characterize the potential microbial activity using 14C-labeled substrates, as well as the presence, abundance, and distribution of specific hydrocarbon degrading genotypes using DNA:DNA hybridization. Enumeration of total microbial abundance using a 16S rDNA universal oligonucleotide probe was compared to traditional enumeration methods. Total culturable populations determined by spread plate analysis ranged from a low of 10(4) to more than 10(6) organisms per gram sediment. Microbial abundance estimated by DNA hybridization studies with 16S rDNA genes ranged from 10(7) to 10(8) organisms per gram sediment. Molecular analysis of aquifer samples using DNA probes targeting genes encoding the degradative enzymes alkane hydroxylase (alkB), catechol 2,3-dioxygenase (nahH), naphthalene dioxygenase (nahA), toluene dioxygenase (todC1C2), toluene monooxygenase (tomA), and xylene monooxygenase (xylA), as well as two probes measuring methanogenic microorganisms, codh (carbon monoxide dehydrogenase) and mcr (methyl coenzyme reductase), revealed that each target gene sequence was present in nearly all 60 samples. The presence of organisms demonstrating the phenotype to degrade BTEX and naphthalene was further supported using mineralization assays with 14C-labeled benzene, toluene, naphthalene, and phenanthrene. Minimal activity occurred during the first 24 hours. After a period of 5-7 days, greater than 40% of the target compounds were mineralized in aquifer sediments.     

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.     

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.     

The Naturally Transformable Marine Bacterium WJT-1C Formally Identified as “Vibrio” Is a Pseudomonad

A marine bacterial isolate, previously identified as Vibrio WJT-1C (ATCC 55351) and used as a model for investigating the process of natural transformation in the marine environment, has been further examined to determine its taxonomic identity. API 20E test strips, phenotypic testing, and flagellar staining had previously assigned the strain to the genus Vibrio, most closely related to V. campbelli. 16S rRNA analysis indicated that WJT-1C was in the Pseudomonas subgroup of the gamma proteobacteria. Bacteriophage typing and natural transformation with chromosomal DNA indicated that it was distinct from previously described marine transforming pseudomonads including Pseudomonas stutzeri strain JM300. The importance and abundance of the Pseudomonas subgroup of the gamma proteobacteria in the environment suggest that these marine strains are well suited as model organisms for describing the process and importance of natural transformation in nature. Received: 19 February 1996 / Accepted: 29 April 1996     

Cloning of the astaxanthin synthase gene from Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and its assignment as a β-carotene 3-hydroxylase/4-ketolase

A gene has been cloned from Xanthophyllomyces dendrorhous by complementation of astaxanthin formation in a β-carotene accumulating mutant. It consists of 3,166 bp and contains 17 introns. For the β-carotene mutant ATCC 96815, a single point mutation in the splicing sequence of intron 8 was found. The resulting improper splicing of the mRNA results in an inactive protein. The cDNA of this β-carotene oxygenase encodes a cytochrome P450 monooxygenase belonging to the 3A subfamily. P450-specific domains were identified including a cytochrome P450 and an oxygen binding motif. Electrons are provided by a cytochrome P450 reductase. Functional characterization of the enzyme by genetic modification of X. dendrorhous demonstrated that this P450 monooxygenase is multifunctional catalyzing all steps from β-carotene to astaxanthin formation by oxygenation of carbon 3 and 4. The reaction sequence is first 4-ketolation of β-carotene followed by 3-hydroxylation. A hydroxylation mechanism at allylic carbon atoms has been proposed for the generation of 4-keto and 3-hydroxy groups at both β-ionone ends.     

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.