Structural Analysis of a Novel Cyclohexylamine Oxidase from Brevibacterium oxydans IH-35A

Cyclohexylamine oxidase (CHAO) is a flavoprotein first described in Brevibacterium oxydans strain IH-35A that carries out the initial step of the degradation of the industrial chemical cyclohexylamine to cyclohexanone. We have cloned and expressed in Escherichia coli the CHAO-encoding gene (chaA) from B. oxydans, purified CHAO and determined the structures of both the holoenzyme form of the enzyme and a product complex with cyclohexanone. CHAO is a 50 kDa monomer with a PHBH fold topology. It belongs to the flavin monooxygenase family of enzymes and exhibits high substrate specificity for alicyclic amines and sec-alkylamines. The overall structure is similar to that of other members of the flavin monooxygenase family, but lacks either of the C- or N-terminal extensions observed in these enzymes. Active site features of the flavin monooxygenase family are conserved in CHAO, including the characteristic aromatic cage. Differences in the orientations of residues of the CHAO aromatic cage result in a substrate-binding site that is more open than those of its structural relatives. Since CHAO has a buried hydrophobic active site with no obvious route for substrates and products, a random acceleration molecular dynamics simulation has been used to identify a potential egress route. The path identified includes an intermediate cavity and requires transient conformation changes in a shielding loop and a residue at the border of the substrate-binding cavity. These results provide a foundation for further studies with CHAO aimed at identifying features determining substrate specificity and for developing the biocatalytic potential of this enzyme.     

Identification of two gene clusters involved in cyclohexanone oxidation in Brevibacterium epidermidis strain HCU

Brevibacterium epidermidis HCU can grow on cyclic ketones and alcohols as a sole carbon source. We have previously reported the identification of two cyclohexanone-induced Bayer-Villiger monooxygenase genes by mRNA differential display. Using the related technique of Out-PCR, we have amplified large DNA fragments flanking the two monooxygenase genes. Two large gene clusters were sequenced. Several ORFs in each gene cluster encoded proteins homologous to cyclohexanol and cyclohexanone oxidation enzymes from Acinetobacter. However, the structure of these two gene clusters differs significantly from that of Acinetobacter, where the complete pathway has been described. To assess activity of these genes, they were cloned and expressed in Escherichia coli. In vivo and in vitro assays enabled us to assign functions to the expressed ORFs. These ORFs included a cyclohexanol dehydrogenase, two different epsilon-caprolactone hydrolases and two 6-hydroxyhexanoate dehydrogenases belonging to different enzyme families. Because this environmental isolate is difficult to manipulate, we cannot determine at this time which cluster is involved in the degradation of cyclohexanone under physiological conditions. However, the original differential display experiments and some of the experiments reported here suggest the involvement of both gene clusters in the oxidation of cyclic ketones.     

Both FMNH2 and FADH2 can be utilized by the dibenzothiophene monooxygenase from a desulfurizing bacterium Mycobacterium goodii X7B

To investigate the flavin utilization by dibenzothiophene monooxygenase (DszC), DszC of a desulfurizing bacterium Mycobacterium goodii X7B was purified from the recombinant Escherichia coli. It was shown to be able to utilize either FMNH₂ or FADH₂ when coupled with a flavin reductase that reduces either FMN or FAD. Sequence analysis indicated that DszC was similar to the C₂ component of p-hydroxyphenylacetate hydroxylase from Acinetobacter baumannii, which can use both FADH₂ and FMNH₂ as substrates. Both flavins at high concentrations could inhibit the activity of DszC due to autocatalytic oxidation of reduced flavins. The results suggest that DszC should be reclassified as an FMNH₂ and FADH₂ both-utilizing monooxygenase component and the flavins should be controlled at properly reduced levels to obtain optimal biodesulfurization results.     

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.     

Functional assembly of camphor converting two-component Baeyer–Villiger monooxygenases with a flavin reductase from E. coli

The major limitation in the synthetic application of two-component Baeyer–Villiger monooxygenases was addressed by identifying the 28-kDa flavin-reductase Fre from Escherichia coli as a suitable supplier of reduced FMN for these enzymes. Coexpression of Fre with either 2,5- or 3,6-diketocamphane monooxygenase from Pseudomonas putida NCIMB 10007 significantly enhanced the conversion of camphor and norcamphor serving as representative ketones. With purified enzymes, full conversion was achieved, while only slight amounts of product were formed in the absence of this flavin reductase. Fusion of the genes of Fre and DKCMOs into single open reading frame constructs resulted in unstable proteins exhibiting flavin reducing, but poor oxygenating activity, which led to overall decreased conversion of camphor.     

Production of Methyl Ketones from Secondary Alcohols by Cell Suspensions of C2 to C4n-Alkane-Grown Bacteria

Nineteen new C₂ to C₄n-alkane-grown cultures were isolated from lake water from Warinanco Park, Linden, N.J., and from lake and soil samples from Bayway Refinery, Linden, N.J. Fifteen known liquid alkane-utilizing cultures were also found to be able to grow on C₂ to C₄n-alkanes. Cell suspensions of these C₂ to C₄n-alkane-grown bacteria oxidized 2-alcohols (2-propanol, 2-butanol, 2-pentanol, and 2-hexanol) to their corresponding methyl ketones. The product methyl ketones accumulated extracellularly. Cells grown on 1-propanol or 2-propanol oxidized both primary and secondary alcohols. In addition, the activity for production of methyl ketones from secondary alcohols was found in cells grown on either alkanes, alcohols, or alkylamines, indicating that the enzyme(s) responsible for this reaction is constitutive. The optimum conditions for in vivo methyl ketone formation from secondary alcohols were compared among selected strains: Brevibacterium sp. strain CRL56, Nocardia paraffinica ATCC 21198, and Pseudomonas fluorescens NRRL B-1244. The rates for the oxidation of secondary alcohols were linear for the first 3 h of incubation. Among secondary alcohols, 2-propanol and 2-butanol were oxidized at the highest rate. A pH around 8.0 to 9.0 was found to be the optimum for acetone or 2-butanone formation from 2-alcohols. The temperature optimum for the production of acetone or 2-butanone from 2-propanol or 2-butanol was rather high at 60°C, indicating that the enzyme involved in the reaction is relatively thermally stable. Metal-chelating agents inhibit the production of methyl ketones, suggesting the involvement of a metal(s) in the oxidation of secondary alcohols. Secondary alcohol dehydrogenase activity was found in the cell-free soluble fraction; this activity requires a cofactor, specifically NAD. Propane monooxygenase activity was also found in the cell-free soluble fraction. It is a nonspecific enzyme catalyzing both terminal and subterminal oxidation of n-alkanes.     

Characterization of 4-Hydroxyphenylacetate 3-Hydroxylase (HpaB) of Escherichia coli as a Reduced Flavin Adenine Dinucleotide-Utilizing Monooxygenase

4-Hydroxyphenylacetate 3-hydroxylase (HpaB and HpaC) of Escherichia coli W has been reported as a two-component flavin adenine dinucleotide (FAD)-dependent monooxygenase that attacks a broad spectrum of phenolic compounds. However, the function of each component in catalysis is unclear. The large component (HpaB) was demonstrated here to be a reduced FAD (FADH₂)-utilizing monooxygenase. When an E. coli flavin reductase (Fre) having no apparent homology with HpaC was used to generate FADH₂ in vitro, HpaB was able to use FADH₂ and O₂ for the oxidation of 4-hydroxyphenylacetate. HpaB also used chemically produced FADH₂ for 4-hydroxyphenylacetate oxidation, further demonstrating that HpaB is an FADH₂-utilizing monooxygenase. FADH₂ generated by Fre was rapidly oxidized by O₂ to form H₂O₂ in the absence of HpaB. When HpaB was included in the reaction mixture without 4-hydroxyphenylacetate, HpaB bound FADH₂ and transitorily protected it from rapid autoxidation by O₂. When 4-hydroxyphenylacetate was also present, HpaB effectively competed with O₂ for FADH₂ utilization, leading to 4-hydroxyphenylacetate oxidation. With sufficient amounts of HpaB in the reaction mixture, FADH₂ produced by Fre was mainly used by HpaB for the oxidation of 4-hydroxyphenylacetate. At low HpaB concentrations, most FADH₂ was autoxidized by O₂, causing uncoupling. However, the coupling of the two enzymes' activities was increased by lowering FAD concentrations in the reaction mixture. A database search revealed that HpaB had sequence similarities to several proteins and gene products involved in biosynthesis and biodegradation in both bacteria and archaea. This is the first report of an FADH₂-utilizing monooxygenase that uses FADH₂ as a substrate rather than as a cofactor.     

Functional expression of Phanerochaete chrysosporium cellobiose dehydrogenase flavin domain in Escherichia coli

Cellobiose dehydrogenase (CDH; EC 1.1.99.18) is an extracellular glycosylated protein composed of two distinct domains, a C-terminal catalytic flavin domain and an N-terminal cytochrome-b-type heme domain, which transfers electrons from the flavin domain to external electron acceptors. The soluble flavin domain of the Phanerochaete chrysosporium CDH was successfully expressed in Escherichia coli. The enzyme showed dye-mediated CDH activity higher than that of the complete CDH, composed of flavin domain and heme domain, prepared using Pichia pastoris as the host microorganism. The ability to conveniently express the recombinant CDH flavin domain in E. coli provides great opportunities for the molecular engineering of the catalytic properties of CDH.     

Microbial Oxidation of Hydrocarbons: Properties of a Soluble Methane Monooxygenase from a Facultative Methane-Utilizing Organism, Methylobacterium sp. Strain CRL-26

Methylobacterium sp. strain CRL-26 grown in a fermentor contained methane monooxygenase activity in soluble fractions. Soluble methane monooxygenase catalyzed the epoxidation/hydroxylation of a variety of hydrocarbons, including terminal alkenes, internal alkenes, substituted alkenes, branched-chain alkenes, alkanes (C₁ to C₈), substituted alkanes, branched-chain alkanes, carbon monoxide, ethers, and cyclic and aromatic compounds. The optimum pH and temperature for the epoxidation of propylene by soluble methane monooxygenase were found to be 7.0 and 40°C, respectively. Among various compounds tested, only NADH₂ or NADPH₂ could act as an electron donor. Formate and NAD⁺ (in the presence of formate dehydrogenase contained in the soluble fraction) or 2-butanol in the presence of NAD⁺ and secondary alcohol dehydrogenase generated the NADH₂ required for the methane monooxygenase. Epoxidation of propylene catalyzed by methane monooxygenase was not inhibited by a range of potential inhibitors, including metal-chelating compounds and potassium cyanide. Sulfhydryl agents and acriflavin inhibited monooxygenase activity. Soluble methane monooxygenase was resolved into three components by ion-exchange chromatography. All three compounds are required for the epoxidation and hydroxylation reactions.     

Design of a secondary alcohol degradation pathway from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> DSM 50106 in an engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

The genes encoding an alcohol dehydrogenase, Baeyer–Villiger monooxygenase and an esterase from P. fluorescens DSM 50106, which seemed to be metabolically connected based on the sequence of the corresponding open reading frames, were cloned into one vector (pABE) and functionally expressed in Escherichia coli. Overall expression levels were quite low, however, using whole cells of E. coli JM109 pABE expressing the three recombinant enzymes, conversion of secondary alcohols (C_n) to the corresponding primary alcohols (C_n−2) and acetic acid via ketone and ester was possible. In this way, 2-decanol was almost completely converted within 20 h at 30°C. Thus, it could be shown that the three enzymes are metabolically connected and that they are most probably involved in alkane degradation via sub-terminal oxidation of the acyclic aliphatic hydrocarbons.     

Cloning, Baeyer-Villiger biooxidations, and structures of the camphor pathway 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-coenzyme A monooxygenase of Pseudomonas putida ATCC 17453

A dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ³-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor by Pseudomonas putida ATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ³-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli and determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP⁺ at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP⁺. A comparison of several crystal forms of OTEMO bound to FAD and NADP⁺ revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ³-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (k_cat/K_m) favors 2-n-hexyl cyclopentanone (4.3 × 10⁵ M⁻¹ s⁻¹) as a substrate, although its affinity (K_m = 32 μM) was lower than that of the CoA-activated substrate (K_m = 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.     

Screening, Nucleotide Sequence, and Biochemical Characterization of an Esterase from Pseudomonas fluorescens with High Activity towards Lactones

A genomic library of Pseudomonas fluorescens DSM 50106 in a λRESIII phage vector was screened in Escherichia coli K-12 for esterase activity by using α-naphthyl acetate and Fast Blue RR. A 3.2-kb DNA fragment was subcloned from an esterase-positive clone and completely sequenced. Esterase EstF1 was encoded by a 999-bp open reading frame (ORF) and exhibited significant amino acid sequence identity with members of the serine hydrolase family. The deduced amino acid sequences of two other C-terminal truncated ORFs exhibited homology to a cyclohexanone monooxygenase and an alkane hydroxylase. However, esterase activity was not induced by growing of P. fluorescens DSM 50106 in the presence of several cyclic ketones. The esterase gene was fused to a His tag and expressed in E. coli. The gene product was purified by zinc ion affinity chromatography and characterized. Detergents had to be added for purification, indicating that the enzyme was membrane bound or membrane associated. The optimum pH of the purified enzyme was 7.5, and the optimum temperature was 43°C. The showed highest purified enzyme activities towards lactones. The activity increased from γ-butyrolactone (18.1 U/mg) to -caprolactone (21.8 U/mg) to δ-valerolactone (36.5 U/mg). The activities towards the aliphatic esters were significantly lower; the only exception was the activity toward ethyl caprylate, which was the preferred substrate.     

Enhanced production of ɛ-caprolactone by overexpression of NADPH-regenerating glucose 6-phosphate dehydrogenase in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> harboring cyclohexanone monooxygenase gene

Whole-cell conversion of cyclohexanone to ɛ-caprolactone was attempted by recombinant Escherichia coli BL21(DE3) expressing cyclohexanone monooxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871. High concentrations of cyclohexanone and ɛ-caprolactone reduced CHMO-mediated bioconversion of cyclohexanone to ɛ-caprolactone in the resting recombinant E. coli cells. Metabolically active cells were employed by adopting a fed-batch culture to improve the production of ɛ-caprolactone from cyclohexanone. A glucose-limited fed-batch Baeyer–Villiger oxidation where a cyclohexanone level was maintained less than 6 g/l resulted in a maximum ɛ-caprolactone concentration of 11.0 g/l. The maximum ɛ-caprolactone concentration was improved further to 15.3 g/l by coexpression of glucose-6-phosphate dehydrogenase, an NADPH-generating enzyme encoded by the zwf gene which corresponded to a 39% enhancement in ɛ-caprolactone concentration compared with the control experiment performed under the same conditions.     

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.     

Enzyme cascade converting cyclohexanol into ε-caprolactone coupled with NADPH recycling using surface displayed alcohol dehydrogenase and cyclohexanone monooxygenase on E. coli

The application of enzymes as biocatalysts in industrial processes has great potential due to their outstanding stereo-, regio- and chemoselectivity. Using autodisplay, enzymes can be immobilized on the cell surface of Gram-negative bacteria such as Escherichia coli. In the present study, the surface display of an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO) on E. coli was investigated. Displaying these enzymes on the surface of E. coli resulted in whole-cell biocatalysts accessible for substrates without further purification. An apparent maximal reaction velocity V_MAX(app) for the oxidation of cyclohexanol with the ADH whole-cell biocatalysts was determined as 59.9 mU ml⁻¹. For the oxidation of cyclohexanone with the CHMO whole-cell biocatalysts a V_MAX(app) of 491 mU ml⁻¹ was obtained. A direct conversion of cyclohexanol to ε-caprolactone, which is a known building block for the valuable biodegradable polymer polycaprolactone, was possible by combining the two whole-cell biocatalysts. Gas chromatography was applied to quantify the yield of ε-caprolactone. 1.12 mM ε-caprolactone was produced using ADH and CHMO displaying whole-cell biocatalysts in a ratio of 1:5 after 4 h in a cell suspension of OD_578nm 10. Furthermore, the reaction cascade as applied provided a self-sufficient regeneration of NADPH for CHMO by the ADH whole-cell biocatalyst.     

Camphor Pathway Redux: Functional Recombinant Expression of 2,5- and 3,6-Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453 with Their Cognate Flavin Reductase Catalyzing Baeyer-Villiger Reactions

Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (−) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (K_m = 32 μM), and it catalyzes the reduction of flavin mononucleotide (FMN) (K_m = 3.6 μM; k_cat = 283 s⁻¹) in preference to flavin adenine dinucleotide (FAD) (K_m = 19 μM; k_cat = 128 s⁻¹). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE_25–1 for 2,5-DKCMO-1 and camE_25–2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE₃₆). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates.     

Cloning,expression and characterization of a Baeyer-Villiger monooxygenase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2440

The gene encoding a Baeyer-Villiger monooxygenase and identified in Pseudomonas putida KT2440 was cloned and functionally expressed in Escherichia coli. The highest yield of soluble protein could be achieved by co-expression of molecular chaperones. In order to determine the substrate specificity, biocatalyses were performed using crude cell extract, growing and resting cells. Examination of aromatic, cyclic and aliphatic ketones revealed a high specificity towards short-chain aliphatic ketones. Interestingly, some open-chain ketones were converted to the alkylacetates, while for others formation of the ester products with oxygen on the other side of the keto group could also be detected yielding the corresponding methyl or ethyl esters.     

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).     

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.