The roles of soluble factors in squalene epoxidation by rat liver microsomes.

Squalene epoxidation by rat liver microsomes requires a supernatant protein factor and an acidic phospholipid in addition to NADPH and molecular oxygen. This study has shown that both the protein factor and the phospholipid lipid are necessary for externally added squalene to bind to the catalytic site on microsomal membranes. The epoxidation of squalene thus bound or biosynthesized $\text{in}\text{situ}$ from mevalonic acid proceeds effectively if the protein factor is present. Thus, the supernatant protein factor seems to play a dual function in both the binding and epoxidation of squalene in the $\text{in}\text{vitro}$ assay system. The phospholipid is not required for the epoxidation of bound squalene.     

Evidence for a rebinding of antheraxanthin to the light-harvesting complex during the epoxidation reaction of the violaxanthin cycle

In the present study, we investigated the epoxidation reaction of the violaxanthin (Vx) cycle in intact cells of Chlorella vulgaris. Our results show that the overall epoxidation is slightly slower in darkness compared to the epoxidation during high light (HL) illumination. The calculation of the rate constants of the two epoxidation steps revealed that, for both conditions, the first epoxidation step from zeaxanthin (Zx) to antheraxanthin (Ax) is faster than the second epoxidation step from Ax to Vx. However, the most noteworthy result of our present study is that Ax, which is transiently formed during the epoxidation reaction, participates in non-photochemical quenching of chlorophyll fluorescence (NPQ). A correlation between NPQ and the de-epoxidized xanthophyll cycle pigments during the time-course of the epoxidation reaction can only be achieved when NPQ is plotted versus the sum of Zx and Ax. The accumulation of significant amounts of Ax during the epoxidation reaction further indicates that Ax-dependent quenching proceeds with a similar efficiency compared to the Zx-mediated NPQ. As the xanthophyll-dependent NPQ relies on the presence of de-epoxidized xanthophylls in the PS II antenna, Ax-dependent NPQ is only possible under the assumption that Ax rebinds to the light-harvesting complex (LHC) II during the epoxidation reaction.     

Phenylbutazone-dependent epoxidation of 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene. A new mechanism for prostaglandin H synthase-catalyzed oxidations

The nonsteroidal anti-inflammatory drug phenylbutazone markedly enhances the hydroperoxide-dependent epoxidation of 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene catalyzed by microsomal and Tween-20 solubilized preparations of prostaglandin H synthase. Furthermore, phenylbutazone radically alters the hydroperoxide specificity of 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene epoxidation. In the absence of phenylbutazone, only allylic hydroperoxides are effective in initiating epoxidation, whereas in the presence of phenylbutazone the reaction can be initiated by t-butyl hydroperoxide, cumene hydroperoxide, and hydrogen peroxide. All effects are dependent on the concentration of phenylbutazone present. The primary event is the oxidation of phenylbutazone by prostaglandin H synthase. This pathway yields a peroxy radical of phenylbutazone which appears to be the epoxidizing agent. This activation of a primary substrate by a peroxidase resulting in metabolism of a secondary substrate is analogous to the halogenation reactions catalyzed by chloroperoxidase. This represents a new class of oxidation reactions catalyzed by prostaglandin H synthase.     

Chemical and Enzymatic Synthetic Methods for Asymmetric Oxidation of the C-C Double Bond

A number of synthetically useful methods for asymmetric oxidation of the C-C double bond are briefly reviewed. This includes chemical asymmetric epoxidation, such as Sharpless, Julia, and Jacobsen epoxidation, asymmetric cis-dihydroxylation of olefins, monooxygenase-catalyzed epoxidation, dioxygenase-catalyzed cis-dihydroxylation of aromatics, and trans-dihydroxylation of C-C double bond catalyzed by a monooxygenase and an epoxide hydrolase. The catalytic system, substrate range, enantioselectivity, synthetic application, and scope and limitation of each method are described.     

Supplementary ultraviolet-B radiation induces a rapid reversal of the diadinoxanthin cycle in the strong light-exposed diatom Phaeodactylum tricornutum

A treatment of the diatom Phaeodactylum tricornutum with high light (HL) in the visible range led to the conversion of diadinoxanthin (Dd) to diatoxanthin (Dt). In a following treatment with HL plus supplementary ultraviolet (UV)-B, the Dt was rapidly epoxidized to Dd. Photosynthesis of the cells was inhibited under HL + UV-B. This is accounted for by direct damage by UV-B and damage because of the UV-B-induced reversal of the Dd cycle and the associated loss of photoprotection. The reversal of the Dd cycle by UV-B was faster in the presence of dithiothreitol, an inhibitor of the Dd de-epoxidase. Our results imply that the reversal of the Dd cycle by HL + UV-B was caused by an increase in the rate of gross Dt epoxidation, whereas the de-epoxidation of Dd was unaffected by UV-B. This is further supported by our finding that the in vitro de-epoxidation activity and the affinity toward the cosubstrate ascorbic acid of the Dd de-epoxidase were both unaffected by UV-B pretreatment of intact cells. We provide evidence that Dt epoxidation is normally down-regulated by a high pH gradient under HL. It is proposed that supplementary UV-B affected the pH gradient across the thylakoid membrane, which disrupted the down-regulation of Dt epoxidation and led to the observed increase in the rate of Dt epoxidation.     

FAD C(4a)-hydroxide stabilized in a naturally fused styrene monooxygenase

StyA2B represents a new class of styrene monooxygenases that integrates flavin-reductase and styrene-epoxidase activities into a single polypeptide. This naturally-occurring fusion protein offers new avenues for studying and engineering biotechnologically relevant enantioselective biochemical epoxidation reactions. Stopped-flow kinetic studies of StyA2B reported here identify reaction intermediates similar to those reported for the separate reductase and epoxidase components of related two-component systems. Our studies identify substrate epoxidation and elimination of water from the FAD C(4a)-hydroxide as rate-limiting steps in the styrene epoxidation reaction. Efforts directed at accelerating these reaction steps are expected to greatly increase catalytic efficiency and the value of StyA2B as biocatalyst.     

The regulation of xanthophyll cycle activity and of non-photochemical fluorescence quenching by two alternative electron flows in the diatoms Phaeodactylum tricornutum and Cyclotella meneghiniana

Intact cells of diatoms are characterized by a rapid diatoxanthin epoxidation during low light periods following high light illumination while epoxidation is severely restricted in phases of complete darkness. The present study shows that rapid diatoxanthin epoxidation is dependent on the availability of the cofactor of diatoxanthin epoxidase, NADPH, which cannot be generated in darkness due to the inactivity of PSI. In the diatom Phaeodactylum tricornutum, NADPH production during low light is dependent on PSII activity, and addition of DCMU consequently abolishes diatoxanthin epoxidation. In contrast to P. tricornutum, DCMU does not affect diatoxanthin epoxidation in Cyclotella meneghiniana, which shows the same rapid epoxidation in low light both in the absence or presence of DCMU. Measurements of the reduction state of the PQ pool and PSI activity indicate that, in the presence of DCMU, NADPH production in C. meneghiniana occurs via alternative electron transport, which includes electron donation from the chloroplast stroma to the PQ pool and, in a second step, from PQ to PSI. Similar electron flow to PQ is also observed during high light illumination of DCMU-treated P. tricornutum cells. In contrast to C. meneghiniana, the electrons are not directed to PSI, but most likely to a plastoquinone oxidase. This chlororespiratory electron transport leads to the establishment of an uncoupler-sensitive proton gradient in the presence of DCMU, which induces diadinoxanthin de-epoxidation and NPQ. In C. meneghiniana, electron flow to the plastoquinone oxidase is restricted, and consequently, diadinoxanthin de-epoxidation and NPQ is not observed after addition of DCMU.     

Selectivity and neuroendocrine regulation of the precursor uptake by pheromone glands from hemolymph in geometrid female moths, which secrete epoxyalkenyl sex pheromones

Macrolepidopteran female moths in families such as Geometridae produce epoxyalkenyl sex pheromones, which are biosynthesized via epoxidation of polyunsaturated hydrocarbons in their pheromone glands. The precursors, however, are expected to be produced outside of the pheromone glands, probably in oenocytes or in the fat body, and transported to the glands via hemolymph. Based on these facts, the selectivity of the epoxidation substrates and of the precursor uptake by pheromone glands was examined with two geometrid species, Hemerophila artilineata and Ascotis selenaria cretacea, using binary mixtures of deuterated precursors and their analogs, which were topically applied to the pheromone glands or injected into the abdomen. GC-MS measurements of pheromone extracts showed equal epoxidation of two polyenes, indicating a low selectivity for both processes, while the epoxidation proceeded at only one double bond specific to each species. This result makes it possible to conclude that the formation of species-specific epoxyalkenyl pheromones results from the rigid formation of polyunsaturated precursors and their epoxidation at a fixed position. Next, the neuroendocrine regulation of these processes was studied with in vivo and in vitro experiments using decapitated females. The epoxy pheromones disappeared completely within 36 h of decapitation, and epoxidation of the injected precursors was not detected in the decapitated females, which restarted the reaction by treatment with a pheromone biosynthesis-activating neuropeptide (PBAN). The precursors topically applied to glands of the decapitated females, however, were converted into epoxy pheromones without PBAN, indicating that this neuropeptide hormone accelerated the precursor uptake by pheromone glands but not the epoxidation already underway in the glands.     

Chemical and Enzymatic Synthetic Methods for Asymmetric Oxidation of the C–C Double Bond

A number of synthetically useful methods for asymmetric oxidation of the C–C double bond are briefly reviewed. This includes chemical asymmetric epoxidation, such as Sharpless, Julia, and Jacobsen epoxidation, asymmetric cis-dihydroxylation of olefins, monooxygenase-catalyzed epoxidation, dioxygenase-catalyzed cis-dihydroxylation of aromatics, and trans-dihydroxylation of C–C double bond catalyzed by a monooxygenase and an epoxide hydrolase. The catalytic system, substrate range, enantioselectivity, synthetic application, and scope and limitation of each method are described.     

The importance of a highly active and DeltapH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae

The present study focuses on the regulation of diatoxanthin (Dtx) epoxidation in the diadinoxanthin (Ddx) cycle containing algae Phaeodactylum tricornutum, Thalassiosira pseudonana, Cyclotella meneghiniana and Prymnesium parvum and its significance for the control of the photosystem II (PS II) antenna function. Our data show that Dtx epoxidase can exhibit extremely high activities when algal cells are transferred from high light (HL) to low light (LL). Under HL conditions, Dtx epoxidation is strongly inhibited by the light-driven proton gradient. Uncoupling of the cells during HL illumination restores the high epoxidation rates observed during LL. In Ddx cycle containing algae, non-photochemical quenching of chlorophyll fluorescence (NPQ) is directly correlated with the Dtx concentration and independent of the presence of the proton gradient. This means that a fast conversion of PS II from the heat dissipating state back to the light-harvesting state can only be realized by an efficient removal of the quenching pigment Dtx. It is proposed that the high Dtx epoxidation rates during LL illumination serve exactly this purpose. The inhibition of Dtx epoxidation by the DeltapH, on the other hand, ensures rapid increases in the Dtx concentration when photoprotection under conditions of HL illumination is required. The regulation of the PS II antenna function in Ddx cycle containing algae is different to that in violaxanthin (Vx) cycle containing plants, where for the zeaxanthin (Zx)-dependent NPQ the presence of a proton gradient is mandatory. In the green alga Chlorella vulgaris conversion of PS II from the heat dissipating state back to the light-harvesting state is controlled by the DeltapH, whose relaxation after a transition from HL to darkness or LL rapidly abolishes the thermal dissipation of excitation energy, including the Zx-dependent NPQ. Due to the inability of Zx to quench fluorescence in the absence of the DeltapH a fast epoxidation of Zx to Vx in LL is not needed and is missing in Chlorella vulgaris.     

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.     

Short-term down-regulation of zeaxanthin epoxidation in Arabidopsis thaliana in response to photo-oxidative stress conditions

The epoxidation of zeaxanthin (Zx) to violaxanthin after exposure to different light stress conditions has been studied in Arabidopsis (Arabidopsis thaliana). Formation of Zx was induced by illumination of intact leaves for up to 8 h at different light intensities and temperatures. The kinetics of epoxidation was found to be gradually retarded with increasing light stress during pre-illumination, indicating a gradual down-regulation of the Zx epoxidase activity. Retardation of the epoxidation rates by a factor of up to 10 was inducible either by increasing the light intensity or by extending the illumination time or by decreasing the temperature during pre-illumination. The retardation of the epoxidation kinetics was correlated with a decrease of the PSII quantum efficiency after the pre-illumination treatment. Experiments with the stn7/stn8 mutant of Arabidopsis indicated that the thylakoid protein kinases STN7 and STN8, which are required for the phosphorylation of PSII proteins, are not involved in the short-term down-regulation of Zx epoxidation. However, the retardation of Zx epoxidation was maintained in thylakoids isolated from pre-illuminated leaves, indicating that a direct modification of the Zx epoxidase is most likely involved in the light-induced down-regulation.     

Utilization of peroxide and its relevance in oxygen insertion reactions catalyzed by chloroperoxidase.

Chloroperoxidase (CPO) catalyzed oxygen insertions are highly enantioselective and hence of immense biotechnological potential. A peroxide activation step is required to give rise to the compound I species that catalyzes this chiral reaction. A side reaction, a catalase type peroxide dismutation, is another feature of CPO's versatility. This work systematically investigates the utilization of different peroxides for the two reactions, i.e. the catalase type reaction and the oxygen insertion reaction. For the oxygen insertion reaction, indene and phenylethyl sulfide were chosen as substrate models for epoxidation and sulfoxidation respectively. The results clearly show that CPO is stable towards hydrogen peroxide and has a total number of turnovers near one million prior to deactivation. The epoxidation reactions terminate before completion because the enzyme functioning in its catalatic mode quickly removes all of the hydrogen peroxide from the reaction mixture. Sulfoxidation reactions are much faster than epoxidation reactions and thus are better able to compete with the catalase reaction for hydrogen peroxide utilization. A preliminary study towards optimizing the reaction system components for a laboratory scale synthetic epoxidation is reported.     

Parallel mechanistic studies on the counterion effect of manganese salen and porphyrin complexes on olefin epoxidation by iodosylarenes

Counterions of manganese(III) porphyrin complexes influence diastereoselectivity in cis-stilbene epoxidation and product distribution in cyclohexene epoxidation markedly. In the epoxidation of cis-stilbene by iodosylbenzene carried out in a solvent mixture of CH(3)CN and CH(2)Cl(2), trans-stilbene oxide is the major product in the reaction of manganese complexes bearing a ligating anion (i.e., Cl(-)), whereas cis-stilbene oxide is the dominant product in the reactions of manganese complexes bearing a poorly-ligating anion (i.e., CF(3)SO(4)(-)). In cyclohexene epoxidation, the yields of allylic oxidation products such as cyclohexenol and cyclohexenone are higher when the counterion of the manganese catalysts is Cl(-) than when the counterion is CF(3)SO(4)(-). The product selectivities are also dependent on the nature of iodosylarenes and the axial and porphyrin ligands of the manganese porphyrin catalysts. The observation that product selectivities are different depending on the iodosylarenes may indicate the involvement of multiple oxidants in oxygen atom transfer reactions. These results are compared with those observed in manganese salen-catalyzed epoxidation of olefins by iodosylarenes.     

Modification of chiral dimethyl tartrate through transesterification: Immobilization on POSS and enantioselectivity reversal in sharpless asymmetric epoxidation

Modification of dimethyl tartrate has been investigated through transesterification with aminoalcohols to provide reactive functionalities for the covalent bonding of chiral tartrate to polyhedral oligomeric silsesquioxanes. The transesterification of dimethyl tartrate has been widely studied using different catalytic systems and reaction conditions. Through the proper selection of both the catalytic system and the reaction conditions, it is possible to achieve monosubstituted or bis‐substituted tartrate derivatives as sole products. All the intermediate chiral tartrate‐derived ligands were successfully used in the homogeneous enantioselective epoxidation of allylic alcohols providing moderate enantiomeric excess over the products. Attached amine groups have been used to support the modified tartrate ligands on to a haloaryl‐functionalized silsesquioxane moiety. This final chiral tartrate ligand displays reverse enantioselectivity in the asymmetric epoxidation of allylic alcohols with regard to the starting dimethyl tartrate ligand, both molecules having the same chiral sign. However, the POSS‐containing ligand can be easily recovered in almost quantitative yield and reused in asymmetric epoxidation reactions. In addition, recovered silsesquioxane‐pendant ligand, though displaying decreasing catalytic activity in recycling epoxidation tests, showed very stable enantioselective behavior. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Amino sugars: new inhibitors of zeaxanthin epoxidase, a violaxanthin cycle enzyme

The effect of three sugars and their amino derivatives on violaxanthin cycle enzymes activity was investigated in duckweed (Lemna trisulca), a model water-plant. No effect of sugars and amino sugars on violaxanthin de-epoxidase was observed independent of incubation time; however, epoxidation of zeaxanthin to violaxanthin was inhibited. The minimum amino sugar concentrations causing maximum inhibition of zeaxanthin epoxidation have been estimated. Amino sugars but not sugars caused more than a 50% inhibition of zeaxanthin epoxidation in duckweed after a 24h incubation when applied at a concentration of 0.5%. Incubation with amino sugars under a 6d photoperiod enhanced the inhibitory effect. Zeaxanthin epoxidation was completely inhibited under such conditions, whereas only a minor inhibitory effect was observed in sugar treated plants. The strong amino sugar inhibition of zeaxanthin epoxidase activity represents additional evidence for the creation of an unstable carotenoid carbocation in the molecular mechanism of epoxidation.     

Short-term down-regulation of zeaxanthin epoxidation in Arabidopsis thaliana in response to photo-oxidative stress conditions

The epoxidation of zeaxanthin (Zx) to violaxanthin after exposure to different light stress conditions has been studied in Arabidopsis (Arabidopsis thaliana). Formation of Zx was induced by illumination of intact leaves for up to 8 h at different light intensities and temperatures. The kinetics of epoxidation was found to be gradually retarded with increasing light stress during pre-illumination, indicating a gradual down-regulation of the Zx epoxidase activity. Retardation of the epoxidation rates by a factor of up to 10 was inducible either by increasing the light intensity or by extending the illumination time or by decreasing the temperature during pre-illumination. The retardation of the epoxidation kinetics was correlated with a decrease of the PSII quantum efficiency after the pre-illumination treatment. Experiments with the stn7/stn8 mutant of Arabidopsis indicated that the thylakoid protein kinases STN7 and STN8, which are required for the phosphorylation of PSII proteins, are not involved in the short-term down-regulation of Zx epoxidation. However, the retardation of Zx epoxidation was maintained in thylakoids isolated from pre-illuminated leaves, indicating that a direct modification of the Zx epoxidase is most likely involved in the light-induced down-regulation.     

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.     

Lipoxygenase-catalyzed epoxidation of benzo(a)pyrene-7,8-dihydrodiol

Metabolism of resolved radioactive stereoisomer, [14C](+)-benzo-(a)pyrene-trans-7,8-dihydrodiol by highly purified soybean lipoxygenase plus linoleic acid was investigated. Trans-anti-7,8,9,10-tetrahydrotetrol, the product of hydrolytic breakdown of ultimate mutagenic benzo(a)pyrene-anti-7,8-dihydrodiol,9,10-epoxide, was detected as a major metabolite. The epoxidation, depended on the enzyme concentration and was inhibited by nordihydroguaiaretic acid. This study provides evidence on the ability of lipoxygenase to catalyze the epoxidation of benzo(a)pyrene-7,8-dihydrodiol.     

Directed evolution of cytochrome P450 for sterol epoxidation

16,17-Epoxysterol plays an important role in pharmaceutical steroid synthesis. To investigate the potential application of cytochrome P450 for epoxysterol synthesis, an approach to the epoxidation of 16,17-epoxysterol, based on directed evolution of cytochrome P450 BM-3, was developed. This comprised random gene mutagenesis for optimizing the activity of P450 BM-3 for epoxidation of hydrophobic sterol, followed by the 7-ethoxycoumarin de-ethylation assay for general enzyme activity detection and the modified picric acid assay for epoxidation activity screening. By the two-step screening, one mutant from 792 clones showed specific substrate activity of converting progesterone to 16,17-epoxysterol, which validated the possibility to evolve the cytochrome P450 for the synthesis of steroidal epoxides.