Enantioselectivity in the Rabbit Liver Microsomal Biotransformation of Styrene and Phenylpropenes

The rabbit liver microsomal P-450 catalyzed oxidation of styrene (1a) and isomeric phenylpropenes, trans-1-phenylpropene (1b), cis-1-phenylpropene (1c) and 3-phenylpropene (1d), was investigated and the enantioselectivity of the epoxidation of the olefinic double bond was determined by checking the enantiomeric excesses of the corresponding first formed epoxides (2). These enantiomeric excesses were always modest, ranging between 7% of (1S,2S)-(2b) and 22% of (1R,2R)-(2c). In the case of (1d) a nonenantioselective hydroxylation at the benzylic-allylic C(3) was also oberved. The ratio between this hydroxylation and olefin epoxidation of (Id) was 1:2.     

Alteration of the stereo- and regioselectivity of alkene monooxygenase based on coupling protein interactions

Alkene monooxygenase from Xanthobacter autotrophicus Py2 (XAMO) catalyses the asymmetric epoxidation of a broad range of alkenes. As well as the electron transfer components (a NADH-oxidoreductase and a Rieske-type ferredoxin) and the terminal oxygenase containing the binuclear non-haem iron active site, it requires a small catalytic coupling/effector protein, AamD. The effect of changing AamD stoichiometry and substitution with effector protein homologues on the regioselectivity of toluene hydroxylation and stereoselectivity of styrene epoxidation has been studied. At sub-optimal stoichiometries, there was a marked change in regioselectivity, but no significant change in epoxidation stereoselectivity. Recombinant coupling proteins from a number of phylogenetically related oxygenases were investigated for their ability to functionally replace AamD. Substitution of AamD with IsoD, the coupling protein from the closely related isoprene monooxygenase, changed the regioselectivity of toluene hydroxylation and stereoselectivity of styrene epoxidation, although this was accompanied by a high level of uncoupling. This indicates the importance of coupling protein interaction in controlling the catalytic specificity. Sequence analysis suggests that interaction between Asn34 and Arg57 is important for complementation specificity of the coupling proteins, providing a candidate for site-directed mutagenesis studies.     

Engineering sequence and selectivity of late-stage C-H oxidation in the MycG iterative cytochrome P450

MycG is a multifunctional P450 monooxygenase that catalyzes sequential hydroxylation and epoxidation or a single epoxidation in mycinamicin biosynthesis. In the mycinamicin-producing strain Micromonospora griseorubida A11725, very low-level accumulation of mycinamicin V generated by the initial C-14 allylic hydroxylation of MycG is observed due to its subsequent epoxidation to generate mycinamicin II, the terminal metabolite in this pathway. Herein, we investigated whether MycG can be engineered for production of the mycinamicin II intermediate as the predominant metabolite. Thus, mycG was subject to random mutagenesis and screening was conducted in Escherichia coli whole-cell assays. This enabled efficient identification of amino acid residues involved in reaction profile alterations, which included MycG R111Q/V358L, W44R, and V135G/E355K with enhanced monohydroxylation to accumulate mycinamicin V. The MycG V135G/E355K mutant generated 40-fold higher levels of mycinamicin V compared to wild-type M. griseorubida A11725. In addition, the E355K mutation showed improved ability to catalyze sequential hydroxylation and epoxidation with minimal mono-epoxidation product mycinamicin I compared to the wild-type enzyme. These approaches demonstrate the ability to selectively coordinate the catalytic activity of multifunctional P450s and efficiently produce the desired compounds.     

The axial ligand effect of oxo-iron porphyrin catalysts. How does chloride compare to thiolate?

We have performed density functional theory calculations on an oxo-iron porphyrin catalyst with chloride as an axial ligand and tested its reactivity toward propene. The reactions proceed via multistate reactivity on competing doublet and quartet spin surfaces. Close-lying epoxidation and hydroxylation mechanisms are identified, whereby in the gas phase the epoxidation reaction is dominant, while in environments with a large dielectric constant the hydroxylation pathways become competitive. By contrast to reactions with thiolate as an axial ligand all low-lying pathways have small ring-closure and rebound barriers, so it is expected that side products and rearrangements will be unlikely with Fe=O(porphyrin)Cl, whereas with Fe=O(porphyrin)SH some side products were predicted. The major differences in the electronic configurations of Fe=O(porphyrin)Cl and Fe=O(porphyrin)SH are due to strong mixing of thiolate orbitals with iron 3d orbitals, a mixing which is much less with chloride as an axial ligand. Predictions of the reactivity of ethylbenzene-h ₁₂ versus ethylbenzene-d ₁₂ are made. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Oxidation of organic compounds by propane monooxygenase ofRhodococcus erythropolis 3/89

The ability of propane monooxygenase ofRhodococcus erythropolis 3/89 to catalyze oxidation of higher liquid alkenes and aromatic hydrocarbons was studied. Optimal conditions of tetradecene epoxidation and benzene hydroxylation were found. Under these conditions, oxidation was shown to be accompanied by a 100% conversion of benzene to phenol.     

Epoxidation of Short-Chain Alkenes by Resting-Cell Suspensions of Propane-Grown Bacteria

Sixteen new cultures of propane-utilizing bacteria were isolated from lake water from Warinanco Park, Linden, N.J. and from lake and soil samples from Bayway Refinery, Linden, N.J. In addition, 19 known cultures obtained from culture collections were also found to be able to grow on propane as the sole carbon and energy source. In addition to their ability to oxidize n-alkanes, resting-cell suspensions of both new cultures and known cultures grown on propane oxidize short-chain alkenes to their corresponding 1,2-epoxides. Among the substrate alkenes, propylene was oxidized at the highest rate. In contrast to the case with methylotrophic bacteria, the product epoxides are further metabolized. Propane and other gaseous n-alkanes inhibit the epoxidation of propylene. The optimum conditions for in vivo epoxidation are described. Results from inhibition studies indicate that a propane monooxygenase system catalyzes both the epoxidation and hydroxylation reactions. Experiments with cell-free extracts show that both hydroxylation and epoxidation activities are located in the soluble fraction obtained after 80,000 × g centrifugation.     

Oxoiron(IV) porphyrin π-cation radical complexes with a chameleon behavior in cytochrome P450 model reactions

There is an intriguing, current controversy on the involvement of multiple oxidizing species in oxygen transfer reactions by cytochromes P450 and iron porphyrin complexes. The primary evidence for the multiple oxidants theory was that products and/or product distributions obtained in the catalytic oxygenations were different depending on reaction conditions such as catalysts, oxidants, and solvents. In the present work, we carried out detailed mechanistic studies on competitive olefin epoxidation, alkane hydroxylation, and C=C epoxidation versus allylic C–H hydroxylation in olefin oxygenation with in situ generated oxoiron(IV) porphyrin -cation radicals (1) under various reaction conditions. We found that the products and product distributions were markedly different depending on the reaction conditions. For example, 1 bearing different axial ligands showed different product selectivities in competitive epoxidations of cis-olefins and trans-olefins and of styrene and para-substituted styrenes. The hydroxylation of ethylbenzene by 1 afforded different products, such as 1-phenylethanol and ethylbenzoquinone, depending on the axial ligands of 1 and substrates. Moreover, the regioselectivity of C=C epoxidation versus C–H hydroxylation in the oxygenation of cyclohexene by 1 changed dramatically depending on the reaction temperatures, the electronic nature of the iron porphyrins, and substrates. These results demonstrate that 1 can exhibit diverse reactivity patterns under different reaction conditions, leading us to propose that the different products and/or product distributions observed in the catalytic oxygenation reactions by iron porphyrin models might not arise from the involvement of multiple oxidizing species but from 1 under different circumstances. This study provides strong evidence that 1 can behave like a chameleon oxidant that changes its reactivity and selectivity under the influence of environmental changes.Electronic Supplementary Material Supplementary material is available for this article at .     

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.     

Alkaloid biosynthesis in somatic hybrids of Duboisia leichhardtii F. Muell. and Nicotiana tabacum L.

Somatic hybrids of Duboisia leichhardtii and Nicotiana tabacum were obtained by electrofusion followed by individual cloning. The hybrid nature of the cloned cells and regenerated shoots was confirmed by cytological investigation and ribosomal-DNA analysis, respectively. The hybrid plantlets predominantly produced nicotine, while Duboisia plantlets produced both tropane and nicotine alkaloids. Activities involved in tropane-alkaloid biosynthesis were examined in a series of precursor-feeding experiments. The presence in the hybrid plants of activities responsible for the reduction of tropinone, the hydroxylation and epoxidation of hyoscyamine, and the conversion of nicotine to nornicotine demonstrated the presence of the Duboisia genes for these enzyme activities.We thank Mr. T. Shikanai, Kyoto University, for the preparation of rice rRNA. We also appreciate Dr. J.H. Fitchen, Kyoto University, for critical discussion and English correction.     

Hydroxylation,Epoxidation, and Dehydrogenation of Capsaicin by a Microbial Promiscuous Cytochrome P450 105D7

Cytochrome P450 enzymes (P450s) are versatile biocatalysts, which insert a molecular oxygen into inactivated C−H bonds under mild conditions. CYP105D7 from Streptomyces avermitilis has been reported as a bacterial substrate-promiscuous P450 which catalyzes the hydroxylation of 1-deoxypentalenic acid, diclofenac, naringenin, compactin and steroids. In this study, CYP105D7 catalyzes hydroxylation, epoxidation and dehydrogenation of capsaicin, a pharmaceutical agent, revealing its functional diversity. The kinetic parameters of the CYP105D7 oxidation of capsaicin were determined as K_m=311.60±87.30 μM and k_cat=2.01±0.33 min⁻¹. In addition, we conducted molecular docking, mutagenesis and substrate binding analysis, indicating that Arg81 plays crucial role in the capsaicin binding and catalysis. To our best knowledge, this study presents the first report to illustrate that capsaicin can be catalyzed by prokaryotic P450s.     

Microbial Transformation of 14‐Anhydrodigoxigenin by Alternaria alternata

The microbial transformation of 14‐anhydrodigoxigenin ( 1 ) by Alternaria alternata CGMCC 3.577 led to the production of seven new metabolites, 2 – 8 . Their structures were determined by extensive spectroscopic (CD, IR, 1D‐ and 2D‐NMR, and HR‐ESI‐MS) data analyses. The reactions in the bioprocess exhibited diversity, including specific oxidation, hydroxylation, reduction, epoxidation, and dehydration. In addition, a hypothetical biocatalytic pathway is proposed.     

Identification and characterization of <Emphasis Type="Italic">gerPI</Emphasis> and <Emphasis Type="Italic">gerPII</Emphasis> involved in epoxidation and hydroxylation of dihydrochalcolactone in <Emphasis Type="Italic">Streptomyces</Emphasis> species KCTC 0041BP

The macrolide antibiotics are biosynthesized by initial assembly of a macrolactone ring, followed by a series of post-polyketide (PKS) modifications. In general, the additional hydroxyl or epoxy groups are installed by cytochrome P450 enzymes, improving the bioactivity profile through structural diversification of natural products. The biosynthetic gene cluster for the 16-membered macrolide antibiotic dihydrochalcomycin (DHC) has been cloned from Streptomyces sp. KCTC 0041BP. Three cytochrome P450 genes are found in the DHC biosynthetic gene (ger) cluster. Two P450 enzymes were characterized from this cluster. Disruption of gerPI accumulated predominantly 12,13-de-epoxydihydrochalcomycin while disruption of gerPII accumulated 8-dehydroxy-12,13-de-epoxydihydrochalcomycin; DHC production was abolished in both cases. The results suggest that GerPII P450 catalyzes hydroxylation at the C₈ position followed by an epoxidation reaction catalyzed by GerPI P450 at the C₁₂–C₁₃ position.     

Cytochrome P450cam catalyzed epoxidation of dehydrocamphor

In addition to the normal 5-$\text{exo}$-hydroxylation of camphor, bacterial cytochrome P450 is shown to carry out the facile epoxidation of dehydrocamphor to give $\text{exo}$-5,6-epoxycamphor. A detailed kinetic study of the reaction demonstrates that epoxidation and hydroxylation reactions occur with nearly identical rates both in the reconstituted system containing flavoprotein dehydrogenase, iron-sulfur protein, and NADH as well as in the single turnover reaction beginning with ferrous, oxygenated cytochrome P450. Dehydrocamphor is not a suicide substrate for the enzyme since competent enzyme remains after several thousand reaction cycles per P450 molecule.     

Concerted response of xanthophyll-cycle pigments in a marine diatom, Chaetoceros gracilis, to shifts in light condition

The interconversion rate of diadinoxanthin (DD) cycle under high irradiance and subsequent darkness was analyzed using the cultivated centric marine diatom, Chaetoceros gracilis Schütt. A prompt de‐epoxidation from diadinoxanthin to diatoxanthin (DT) occurred immediately after the onset of higher irradiance. The fist‐order rate constant, k, for this de‐epoxidation was 0.1–0.2 min‐¹ irrespective of the irradiance. The difference in photon fluence rate lead to the difference of the final amount of DT, leaving the rate constant at almost the same value. After turning off the light, epoxidation from DT to DD occurred. The first‐order constant of epoxidation was much slower than that of de‐epoxidation: 0.005 – 0.009 min‐¹. Independent of this epoxidation process, de novo synthesis of DD‐cycle pigments was also observed under the subsequent darkness. Based on these findings, a common nature of the DD‐cycle as a protection mechanism for photo‐systems was demonstrated for C. gracilis.     

Characterization of the ω-hydroxylase of Pseudomonas oleovorans as a nonheme iron protein

The ω-hydroxylase of Pseudomonas oleovorans, which catalyzes the hydroxylation of fatty acids and alkanes and the epoxidation of alkenes in the presence of a reduced pyridine nucleotide, a reductase, rubredoxin, and molecular oxygen, has been purified to electrophoretic homogeneity. Octane hydroxylation and octadiene epoxidation activities appear to remain at a constant ratio during the purification procedure. The hydroxylase has been characterized as a nonheme iron protein containing one iron atom and one cysteine residue per polypeptide chain of molecular weight 40,800. The enzyme is inhibited by cyanide, and activity is restored upon removal of the cyanide by dialysis. Iron is removed from the enzyme by dialysis against EDTA provided that a reducing agent such as dithionite or ascorbate is also added, and enzyme activity is restored by the addition of ferrous ions to the apohydroxylase.     

Assimilation of propane and properties of propan monooxygenase from Rhodococcus erythropolis 3/89

The ability of propane-assimilating microorganisms of the genus Rhodococcus to utilize metabolites of the terminal and subterminal pathways of propane oxidation was studied. Propane monooxygenase of Rhodococcus erythropolis 3/89 was shown to be the an inducible enzyme catalyzing epoxidation and hydroxylation of organic compounds. The optimum conditions for epoxidation of gaseous and liquid alkenes and hydroxylation of aromatic carbohydrates were found.     

Biocatalytic synthesis of C3 chiral building blocks by chloroperoxidase‐catalyzed enantioselective halo‐hydroxylation and epoxidation in the presence of ionic liquids

The optically active C3 synthetic blocks are remarkably versatile intermediates for the synthesis of numerous pharmaceuticals and agrochemicals. This work provides a simple and efficient enzymatic synthetic route for the environment‐friendly synthesis of C3 chiral building blocks. Chloroperoxidase (CPO)‐catalyzed enantioselective halo‐hydroxylation and epoxidation of chloropropene and allyl alcohol was employed to prepare C3 chiral building blocks in this work, including (R)‐2,3‐dichloro‐1‐propanol (DCP*), (R)‐2,3‐epoxy‐1‐propanol (GLD*), and (R)‐3‐chloro‐1‐2‐propanediol (CPD*). The ee values of the formed C3 chiral building blocks DCP*, CPD*, and glycidol were 98.1, 97.5, and 96.7%, respectively. Moreover, the use of small amount of imidazolium ionic liquid enhanced the yield efficiently due to the increase of solubility of hydrophobic organic substrates in aqueous reaction media, as well as the improvement of affinity and selectivity of CPO to substrate. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:724–729, 2015     

Hydroxylation of daidzein by CYP107H1 from Bacillus subtilis 168

CYP107H1, from Bacillus subtilis 168 known as fatty acid hydroxylase, showed the ortho-specific hydroxylation activity to daidzein, when coupled to the putidaredoxin reductase (camA) and putidaredoxin (camB) from Pseudomonas putida as the redox partners. The electron transfer system of the three proteins was constructed in Escherichia coli BL21 (DE3) system using the two plasmids containing different selection markers. The daidzein hydroxylation was demonstrated with recombinant whole cell and in vitro system using the artificial redox partner for electron transfer. The identification of the hydroxylation reaction yielding 7,3′,4′-trihydroxyisoflavone was elucidated using gas chromatography mass spectrometry (GC–MS). This oxidizing activity of CYP107H1 towards daidzein represents the new hydroxylation of aromatic compound as substrate.     

Microbial oxidation of gaseous hydrocarbons. II. Hydroxylation of alkanes and epoxidation of alkenes by cell-free particulate fractions of methane-utilizing bacteria.

Cell-free particulate fractions derived from methylotrophic bacteria catalyze the oxygen- and reduced nicotinamide adenine dinucleotide-dependent epoxidation of alkenes and hydroxylation of alkanes. Evidence presented indicates that the hydroxylation and epoxidation reactions are catalyzed by the same or a similar metal-containing monooxygenase.     

Enhanced hydroxylation of imidacloprid by <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> upon addition of sucrose

Sucrose’s ability to promote the hydroxylation of imidacloprid (IMI) by bacterium Stenotrophomonas maltophilia strain CGMCC 1.1788 was examined. Both growing culture and resting cells could transform IMI into 5-hydroxy IMI. Adding 2% sucrose to the growing culture transformation broth and 5% sucrose to the resting cell transformation broth resulted in biotransformation yields, respectively, 2.5 and 9 times greater than without sucrose. In the growing culture transformation, sucrose increased biomass, which led to enhance hydroxylation of IMI. In the resting cell transformation, sucrose was used not as a carbon source but as an energy source for cofactor regeneration for hydroxylation of IMI. The hydroxylation activity of IMI was promoted eightfold by adding reduced nicotinamide adenine dinucleotide (NADH) to the cell-free extract. The hydroxylation of IMI was significantly inhibited by P450 inhibitor piperonyl butoxide. It seems that the hydroxylation of IMI by S. maltophilia CGMCC 1.1788 might proceed through a system by cooperating with P450 enzyme.