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.     

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.     

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.     

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.     

Enantiospecific Production of S-(-)-2-Hydroxypropiophenone Mediated by Benzoylformate Decarboxylase from Acinetobacter Calcoaceticus

Whole cells of Acinetobacter calcoaceticus, grown in a medium containing mandelate, converted benzoylformate and acetaldehyde into the acyloin compound 2-hydroxypropiophenone. The optical purity of the product was found to be greater than 98%. The absolute configuration of the biotransformation product at the carbinol carbon was found to be (S). The enzyme responsible for this bioconversion was confirmed as benzoylformate decarboxylase by the demonstration that the purified homogeneous enzyme catalysed the condensation reaction.     

A method for rapid screening of ketone biotransformations: detection of whole cell Baeyer-Villiger monooxygenase activity

A method for screening of ketone biotransformations was developed and applied to the identification of Baeyer-Villiger monooxygenase (BVMO) activity. The method was based on the formation of a purple coloured product on reaction between an enolizable ketone and 3,5-dinitrobenzoic acid in an alkaline solution. Absorbance of the colour decreased with the size of the cycloketone ring. Stoichiometric ratio between cycloketone and 3,5-dinitrobenzoic acid was 1:1 at maximum absorbance. The method was applied for monitoring the consumption of cyclohexanone by bacteria under aerobic conditions, and was found to be potentially useful for both screening assays and quantitative measurements of BVMO activity. Compared to other existing methods, this method is faster, cheaper and amenable for whole cell assays.     

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.     

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,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.     

Directed evolution of a Baeyer–Villiger monooxygenase to enhance enantioselectivity

The Baeyer-Villiger monooxygenase (BVMO) BmoF1 from Pseudomonas fluorescens DSM 50106 was shown before to enantioselectively oxidize different 4-hydroxy-2-ketones to the corresponding hydroxyalkyl acetates, being the first example of a BVMO-catalyzed kinetic resolution of aliphatic acyclic ketones. However, the wild-type enzyme exhibited only moderate E values (E approximately 55). Thus, the enantioselectivity was enhanced by means of directed evolution and optimization of reaction conditions since it was found that higher E values (E approximately 70 for wild-type BmoF1) could already be obtained when performing biotransformations in shake flasks rather than small tubes. In a first step, random mutations were introduced by error-prone polymerase chain reaction, and BmoF1 mutants (>3,500 clones) were screened for improved activity and enantioselectivity using a microtiter-plate-based screening method. Mutations S136L and L252Q were found to increase conversion compared to wild type, while several mutations (H51L, F225Y, S305C, and E308V) were identified enhancing the enantioselectivity to a varying extent (E approximately 75-90). In a second step, beneficial mutations were recombined by consecutive cycles of QuikChange site-directed mutagenesis resulting in a double mutant (H51L/S136L) showing both improved conversion and enantioselectivity (E approximately 86).     

Biotechnological production of chiral organic sulfoxides: current state and perspectives

Chiral organic sulfoxides (COSs) are important compounds that act as chiral auxiliaries in numerous asymmetric reactions and as intermediates in chiral drug synthesis. In addition to their optical resolution, stereoselective oxidation of sulfides can be used for COS production. This reaction is facilitated by oxygenases and peroxidases from various microbial resources. To meet the current demand for esomeprazole, a proton pump inhibitor used in the treatment of gastric-acid-related disorders, and the (S)-isomer of an organic sulfoxide compound, omeprazole, a successful biotechnological production method using a Baeyer-Villiger monooxygenase (BVMO), was developed. In this review, we summarize the recent advancements in COS production using biocatalysts, including enzyme identification, protein engineering, and process development.     

Baeyer-Villiger oxidation of DHEA, pregnenolone, and androstenedione by Penicillium lilacinum AM111

The Baeyer-Villiger monooxygenase (BVMO) produced by Penicillium lilacinum AM111, in contrast to other enzymes of this group known in the literature, is able to process 3beta-hydroxy-5-ene steroid substrates. Transformation of DHEA and pregnenolone yielded, as a sole or main product, 3beta-hydroxy-17a-oxa-d-homo-androst-5-en-17-one, a new metabolite of these substrates; pregnenolone was transformed also to testololactone. Testololactone was the only product of oxidation of androstenedione by P. lilacinum AM111. Investigations of the time evolution of reaction progress have indicated that the substrates stimulate activity of BVMO(s) of P. lilacinum AM111.     

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.     

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.     

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.     

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.     

Oxidative biotransformations by microorganisms: production of chiral synthons by cyclopentanone monooxygenase fromPseudomonas sp. NCIMB 9872

Cyclopentanone monooxygenase, an NADPH- plus FAD-dependent enzyme induced by the growth ofPseudomonas sp. NCIMB 9872 on cyclopentanol, has been utilised as a biocatalyst in Baeyer-Villiger oxidations. Washed whole-cell preparations of the microorganism oxidised 3-hexylcyclopentanone in a regio- but not enantioselective manner to give predominantly the racemic γ-hexyl valerolactone. similar preparations biotransformed 5-hexylcyclopent-2-enone exclusively by regio- plus enantioselective oxidation to the equivalent , β-unsaturated (S)-(+)-δ-hexyl valerolactone (ee = 78%), with no reductive biotransformations catalysed by either EC 1.1.x.x- or EC 1.3.x.x-type dehydrogenases.

An equivalent biotransformation of 5-hexylcyclopent-2-enone was catalysed by highly-purified NADPH- plus FAD-dependent cyclopentanone monooxygenase from the bacterium. The regio- plus enantioselective biotransformation by the pure enzyme of 2-(2′-acetoxyethyl)cyclohexanone yielded optically-enriched (S)-(+ )-7-(2′-acetoxyethyl)-2-oxepanone (ee = 72%). The same biotransformation when scaled up again provided optically-enriched (S)-(+)-ε-caprolactone which was converted, using methoxide, to (S)-(−)-methyl 6,8-dihydroxyoctanoate (ee = 42%). thereby providing a two-step access from the substituted cyclohexanone to this important chiron for the subsequent synthesis of (R-(+)-lipoic acid.

Some characteristics of pure NADPH- plus FAD-dependent cyclopentanone monooxygenase were determined including the molecular weight of the monomeric subunit (50000) of this homotetrameric enzyme, and the N-terminal amino acid sequence up to residue 29, which includes a putative flavin nucleotide-binding site.     

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.     

Baeyer-Villiger单加氧酶非保守Hinge影响酶的催化活性和立体选择性

Baeyer-Villiger单加氧酶是一种重要的生物催化剂,可用于合成一系列有价值的酯和内酯化合物。通过序列比对和晶体结构分析推测连接NADPH结构域和FAD结构域的一段非保守Hinge可能在酶对底物识别和催化氧化过程中扮演着重要角色。在以环己酮单加氧酶为模型的研究中发现,对该Hinge结构进行同源序列替换得到的突变体几乎完全丧失了催化活性,证明了其整体水平的重要性。丙氨酸扫描突变揭示其中一些位点对酶的功能有显著影响:K153位点的改变使酶的活性下降,立体选择性却更优化;L143位点的改变对酶的活性影响较小,却降低了立体选择性;L144位点的改变则同时大幅度削弱酶的活性和立体选择性。将同样的方法运用在苯丙酮单加氧酶中,我们得到了相似的结论,证明这些位点的重要功能在Baeyer-Villiger单加氧酶家族中有一定的普遍性。这一研究增进了对Baeyer-Villiger单加氧酶的结构与功能关系的认识,有助于底物结合口袋的精确描述和Baeyer-Villiger单加氧酶催化图景的进一步细化,对未来相关的理性设计和定向改造研究提供了借鉴。