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.     

Expanding the set of rhodococcal Baeyer–Villiger monooxygenases by high-throughput cloning, expression and substrate screening

To expand the available set of Baeyer-Villiger monooxygenases (BVMOs), we have created expression constructs for producing 22 Type I BVMOs that are present in the genome of Rhodococcus jostii RHA1. Each BVMO has been probed with a large panel of potential substrates. Except for testing their substrate acceptance, also the enantioselectivity of some selected BVMOs was studied. The results provide insight into the biocatalytic potential of this collection of BVMOs and expand the biocatalytic repertoire known for BVMOs. This study also sheds light on the catalytic capacity of this large set of BVMOs that is present in this specific actinomycete. Furthermore, a comparative sequence analysis revealed a new BVMO-typifying sequence motif. This motif represents a useful tool for effective future genome mining efforts.     

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.     

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.     

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.     

Screening,expression, and characterization of Baeyer-Villiger monooxygenases for the production of 9-(nonanoyloxy)nonanoic acid from oleic acid

In this study, the production of 9-(nonanoyloxy) nonanoic acid from oleic acid was investigated. The whole cell biotransformation of oleic acid includes OhyA (hydratase), ADH (alcohol dehydrdogenase), and BVMO (Baeyer-Villiger Monooxygenase) enzymes consecutively. BVMOs are known to catalyze oxidative cleavage of long chain aliphatic ketones (e.g., 2-decanone, 10-ketooctadecanoic acid). However, the enzymes are difficult to overexpress in a soluble form in microorganisms. Thereby, this study has focused on screening and functional expression of the BVMOs in Escherichia coli. Initially BVMOs were selected by protein sequence analysis and were examined for their ability to express in soluble and active form to generate 9-(nonanoyloxy)nonanoic acid from oleic acid. Secondly various optimization strategies of inducer concentrations, co-expression with molecular chaperones, and different media conditions were investigated. Among the 9 BVMOs screened, three BVMOs were found to produce the target product and among these, Di_BVMO3 isolated from Dietzia sp. D5 was found to be best. Further, the soluble expression of Di_BVMO3 was enhanced by adding phosphoglycerate kinase as N-terminal fusion tag. The whole cell biotransformation with fusion enzyme resulted in 3 ~ 5-fold enhancement in product formation compared with the non-fusion counterpart. Final productivity up to 105.3 mg/L was achieved. Besides Di-BVMO3, other two new BVMOs of Rh_BVMO4 from Rhodococcus sp. RHA1 and AFL838 from Aspergillus flavus NRRL3357 were screened for production of 9-(nonanoyloxy)nonanoic acid and could be used for whole cell biotransformation reaction of other long chain ketones.     

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.     

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。     

Discovery of Baeyer–Villiger monooxygenases from photosynthetic eukaryotes

Baeyer–Villiger monooxygenases are attractive “green” catalysts able to produce chiral esters or lactones starting from ketones. They can act as natural equivalents of peroxyacids that are the catalysts classically used in the organic synthesis reactions, consisting in the cleavage of CC bonds with the concomitant insertion of an oxygen atom.In this study, two type I BVMOs have been identified for the first time in photosynthetic eukaryotic organisms, the red alga Cyanidioschyzon merolae (Cm) and the moss Physcomitrella patens (Pp). A biocatalytic characterization of these newly discovered enzymes, expressed in recombinant forms, was carried out. Both enzymes could be purified as holo enzymes containing a FAD cofactor. Their thermostability was investigated and revealed that the Cm-BVMO is the most thermostable type I BVMO with an apparent melting temperature of 56 °C. Substrate profiling revealed that both eukaryotic BVMOs accept a wide range of ketones which include aromatic, aliphatic, aryl aliphatic and bicyclic ketones. In particular, linear aliphatic ketones (C9 and C12), carrying the keto functionality in different positions, resulted to be the best substrates in steady state kinetic analyses. In order to restore the BVMO-typifying sequence motif in the Pp-BVMO, a mutant was prepared (Y160H). Intriguingly, this mutation resulted in higher activities on most tested substrates. The recombinant enzymes displayed k_cat values in the 0.1–0.2 s⁻¹ range, which is relatively low when compared with other known type I BVMOs. This may hint to a role in secondary metabolism in these photosynthetic organisms, though their exact function remains to be established.     

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.     

Kinetic and mechanistic properties of biotin sulfoxide reductase

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase catalyzes the reduction of d-biotin d-sulfoxide (BSO) to biotin. Initial rate studies of the homogeneous recombinant enzyme, expressed in Escherichia coli, have demonstrated that the purified protein utilizes NADPH as a facile electron donor in the absence of any additional auxiliary proteins. We have previously shown [Pollock, V. V., and Barber, M. J. (1997) J. Biol. Chem. 272, 3355-3362] that, at pH 8 and in the presence of saturating concentrations of BSO, the enzyme exhibits, a marked preference for NADPH (k(cat,app) = 500 s(-1), K(m,app) = 269 microM, and k(cat,app)/K(m,app) = 1.86 x 10(6) M(-1) s(-1)) compared to NADH (k(cat,app) = 47 s(-1), K(m,app) = 394 microM, and k(cat,app)/K(m,app) = 1.19 x 10(5) M(-1) s(-1)). Production of biotin using NADPH as the electron donor was confirmed by both the disk biological assay and by reversed-phase HPLC analysis of the reaction products. The purified enzyme also utilized ferricyanide as an artificial electron acceptor, which effectively suppressed biotin sulfoxide reduction and biotin formation. Analysis of the enzyme isolated from tungsten-grown cells yielded decreased reduced methyl viologen:BSO reductase, NADPH:BSO reductase, and NADPH:FR activities, confirming that Mo is required for all activities. Kinetic analyses of substrate inhibition profiles revealed that the enzyme followed a Ping Pong Bi-Bi mechanism with both NADPH and BSO exhibiting double competitive substrate inhibition. Replots of the 1/v-axes intercepts of the parallel asymptotes obtained at several low concentrations of fixed substrate yielded a K(m) for BSO of 714 and 65 microM for NADPH. In contrast, utilizing NADH as an electron donor, the replots yielded a K(m) for BSO of 132 microM and 1.25 mM for NADH. Slope replots of data obtained at high concentrations of BSO yielded a K(i) for BSO of 6.10 mM and 900 microM for NADPH. Kinetic isotope studies utilizing stereospecifically deuterated NADPD indicated that BSO reductase uses specifically the 4R-hydrogen of the nicotinamide ring. Cyanide inhibited NADPH:BSO and NADPH:FR activities in a reversible manner while diethylpyrocarbonate treatment resulted in complete irreversible inactivation of the enzyme concomitant with molybdenum cofactor release, indicating that histidine residues are involved in cofactor-binding.     

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

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

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.     

Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca

Type I Baeyer–Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with NADP⁺, we identified four residues that could interact with the 2′-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2′-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant and beneficial effect on the enantioselectivity of Baeyer–Villiger oxidations and sulfoxidations. In conclusion, our results indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex catalytic mechanism and the vital role of the coenzyme in BVMOs.     

Assay optimization and kinetic profile of the human and the rabbit isoforms of 11beta-HSD1

Assay conditions for the 11beta-hydroxysteroid dehydrogenase have been optimized by adding phospholipids in the media buffer to increase and stabilize the enzymatic activity. The presence of phospholipids greatly facilitates the study of the binding of cortisone and NADPH at the enzyme catalytic site. Kinetic analyses conducted with the human and rabbit enzyme isoforms suggest that both enzymes behave according to an ordered sequential bi-bi mechanism where the NADPH is the first to bind at the active site followed by cortisone. The equilibrium dissociation constant, K(i)a as well as the apparent Michaelis-Menten constants K(m)a, K(m)b, k(cat)a, and k(cat)b for NADPH and cortisone, have been determined to be 147.5 microM, 14.4 microM, 43.8 nM, 0.21 min(-1), and 0.27 min(-1), respectively, for the human enzyme and 41.1 microM, 3.1 microM, 161.7 nM, 0.49 min(-1), and 0.52min(-1), respectively, for the rabbit 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).