Accumulation Of Epoxides Research Articles

A tabersonine 3-reductase Catharanthus roseus mutant accumulates vindoline pathway intermediates.

Monoterpenoid indole alkaloids (MIAs) have remarkable biological properties that have led to their medical uses for a variety of human diseases. Mutagenesis has been used to generate plants with new alkaloid profiles and a useful screen for rapid comparison of MIA profiles is described. The MIA mutants identified are useful for investigating MIA biosynthesis and for targeted production of these specialised metabolites. The Madagascar periwinkle (Catharanthus roseus) is the sole source of the dimeric anticancer monoterpenoid indole alkaloids (MIAs), 3',4'-anhydrovinblastine and derivatives, which are formed via the coupling of the MIAs, catharanthine and vindoline. While intense efforts to identify parts of the complex pathways involved in the assembly of these dimers have been successful, our understanding of MIA biochemistry in C. roseus remains limited. A simple thin layer chromatography screen of 4000 ethyl methanesulfonate-metagenized M2 plants is described to identify mutant lines with altered MIA profiles. One mutant (M2-1865) accumulated reduced levels of vindoline inside the leaves in favour of high levels of tabersonine-2,3-epoxide and 16-methoxytabersonine-2,3-epoxide on the leaf surface. This MIA profile suggested that changes in tabersonine 3-reductase (T3R) activity might be responsible for the observed phenotype. Molecular cloning of mutant and wild type T3R revealed two nucleotide substitutions at cytosine residues 565 (CAT to TAT) and 903 (ACC to ACA) in the mutant corresponding to substitution (H189Y) and silent (T305T) amino acid mutations, respectively, in the protein. The single amino acid substitution in the mutant T3R protein diminished the biochemical activity of T3R by 95% that explained the reason for the low vindoline phenotype of the mutant. This phenotype was recessive and exhibited standard Mendelian single-gene inheritance. The stable formation and accumulation of epoxides in the M2-1865 mutant provides a dependable biological source of these two MIAs.

Examining the epoxidation process of soybean oil by peracetic acid

The main principle of green chemistry is the use of renewable, ecological raw materials, which will contribute to subsequent biodegradation and reduction of toxicity of the product in the production of polymers. Vegetable oil (VO) is the cheapest and most common biological raw material, the use of which has such advantages as low toxicity and natural biodegradation. We analyzed experimental dependences of the consumption of reagents and the accumulation of epoxide in the interaction between a solution of soybean oil (SO) in toluene and the epoxidizing systems H 2 O 2 /acetic acid (AA)/KU-2´8 and H 2 O 2 /acetic anhydride (AAn)/KU-2´8. It was established that the use in the process of epoxidation of soybean oil of the specified systems makes it possible to achieve high values of selectivity of epoxidation by double bonds. The resulting values of selectivities in the epoxidation process by double bonds and by the consumption of peroxide when studying the epoxidizing system Н 2 О 2 /AAn/KU-2´8 are higher. The advantages of using the specified epoxidizing system include a reduction in the total volume and mass of the reaction mixture. Obtaining the epoxidized soybean oil with a low resulting value of bromine number provides subsequent good thermal and oxidative stability of materials on its base. We calculated the values of rate constants of the epoxidation reaction of SO at different temperatures. By using the methods of IR and Raman spectroscopic studies, we demonstrated structural changes in raw materials and confirmed the progress of the epoxidation reaction. The developed technique for recalculating the values of bromine, iodine numbers of products of the epoxidation reaction, unsaturation and epoxy number, selectivity of the process in the epoxidation of mixtures of unsaturated compounds allows comparing the results of research. The use of the specified technique also makes it possible to draw unambiguous comparative conclusions about the effectiveness of reagents consumption and the selectivity of reaction. In this case, there is a possibility to improve the technology of obtaining the epoxidized compounds. The calculation formulas obtained were applied to analyze the progress of the epoxidation process of soybean oil.

Mechanism controlling the extended lag period associated with vinyl chloride starvation in Nocardioides sp. strain JS614

The extended lag period associated with vinyl chloride (VC) starvation in VC- and ethene-assimilating Nocardioides sp. strain JS614 was examined. The extended lag periods were variable (3-7 days), only associated with growth on VC or ethene, and were observed in VC- or ethene-grown cultures following 24 h carbon starvation and mid-exponential phase cultures grown on non-alkene carbon sources (e.g. acetate). Alkene monooxygenase (AkMO) and epoxyalkane:coenzyme M transferase (EaCoMT) are the initial enzymes of VC and ethene biodegradation in strain JS614. Reverse-transcription PCR confirmed that the AkMO gene etnC was expressed in response to epoxyethane, a metabolic intermediate of ethene biodegradation. Epoxyethane (0.5 mM) eliminated the extended lag period in both starved and mid-exponential phase cultures, suggesting that epoxyethane accumulation activates AkMO expression in strain JS614. AkMO activity in ethene-grown cultures was not detected after 6.7 h of carbon starvation, while 40% of the initial EaCoMT activity remained after 24 h. Acetate eliminated the extended lag period in starved cultures but not in mid-exponential phase cultures suggesting that acetate reactivates extant AkMO in starved VC- or ethene-grown cultures. The imbalance between AkMO and EaCoMT activities during starvation likely contributes to the extended lag period by delaying epoxide accumulation and subsequent AkMO induction.

Salicylate and methyl jasmonate differentially influence diacetylene accumulation pattern in transformed roots of feverfew

In hairy root cultures of feverfew ( Tanacetum parthenium (L.) Sch. Bip.), treated with methyl jasmonate (MJ), accumulation of spiroketal enol ether type diacetylenes of known deterrent activity was increased about two-fold. The phenomenon was independent of the root clone studied. The maximum total acetylene content (≈2.5 and 1.1% dry weight in clones M2 and I2, respectively) was observed after 72–96 h MJ treatment. Salicylic acid (SA) transiently reduced a content of constitutive spiroketal enol ethers, common for all root clones, but selectively enhanced accumulation of cis-C 13-spiroketal enol ether epoxide (( E)-3,4-epoxy-2-(2,4-hexadiynylidene)-1,6-dioxaspiro[4.4]nonane) ( 2), present in clone M2. Simultaneous addition of SA and MJ to the culture medium influenced diacetylene spectrum in a salicylate-like manner, but accumulation of the epoxide in clone M2 was more abundant (up to 6-fold increase compared with the control).

Tissue distribution of selective warfarin binding sites in the rat

Liver microsomes contain a specific warfarin binding site that is related to the target enzyme vitamin KO reductase [Thijssen HHW and Baars LGM, Biochem Pharmacol 38: 1115–1120, 1989]. In this study the distribution of the warfarin binder in the rat was investigated. Rats were given tracer doses of [ 14C]warfarin and tissue distribution was estimated after a time period. The selectivity of the distribution was verified by the ability of unlabeled warfarin to displace in vivo the tissue accumulated [ 14C]warfarin. The relation to the target enzyme vitamin KO reductase was verified by comparing the results with distribution behavior in the Scottish warfarin-resistant rat strain. The results show that in addition to liver various non-hepatic tissues accumulate warfarin. Among the tissues having a high accumulation ratio and a high rate of exchange by unlabeled warfarin are liver, pancreas, kidney, and salivary gland. Also arteria (aorta), bone, lung and spleen show exchangable [ 14C]warfarin accumulation. In HS rats the [ 14C]warfarin distribution was affected similarly for all tissues; lower levels of accumulation and higher rates of exchange by unlabeled warfarin. The tissue-bound warfarin was recovered predominantly in the microsomal fraction. Its release could only be accomplished in the presence of dithiothreitol and appeared to be stereoselective. The in vivo distribution pattern correlated with the number of warfarin binding sites in the tissue microsomes. The microsomal vitamin KO reductase activity did not always correlate to the binding capacity. The distribution was not affected by vitamin K deficiency. Warfarin-treated rats showed vitamin K epoxide accumulation in most of the organs having the warfarin binder.

Elicitor induction of S-adenosyl-L-methionine: Anthranilic acid N-methyltransferase activity in cell suspension and organ cultures of Ruta graveolens L.

Suspension-cultured cells of Ruta graveolens respond with rapid accumulation of acridone epoxides when treated with autoclaved culture homogenate of Rhodotorula rubra. Here the effect of elicitation on S-adenosyl-L-methionine:anthranilic acid N-methyltransferase (NMT), the first specific step in acridone alkaloid biosynthesis, was investigated. In dark-grown and in light-grown cultures, NMT activity remained at low levels (0.1–1 μkat kg-1 protein) over the entire culturing period of 35 days. Elicitation caused a rapid transient increase of NMT activity up to levels of 9–15 μkat kg-1 for light-grown cultures and 16–22 μkat kg-1 for dark-grown ones. Correlation of elicitor dose and induced NMT activity showed saturation rather than an optimum. Culture age and elicitor concentration strongly affected the return of NMT activity to the constitutive level. Addition of inhibitors of transcription or translation suppressed induction of both NMT activity and acridone epoxide accumulation by 50% (actinomycin) and 90–100% (cycloheximide), respectively. Of all the different suspension and organ cultures tested, roots, the site of high constitutive acridone epoxide accumulation, possessed the highest constitutive NMT activities. Different suspension cultures lines, shoot teratomas, hairy root cultures, and roots and shoots of hydroponically and sterile grown plants all showed increased NMT activity upon elicitation. Roots, however, showed the weakest response and, in contrast to all the other systems, induction was not followed by an increase in acridone epoxide content. Tissue-specific acridone epoxides may function as constitutive metabolites or as phytoalexins.

Elicitor-Induced Accumulation of Acridone Alkaloid Epoxides inRuta graveolensSuspension Cultures

Light and dark grown suspension cultures of RUTA GRAVEOLENS produced small amounts (1-50 microg/g dry weight) of the antimicrobial alkaloids rutacridone epoxide and hydroxyrutacridone epoxide. Acrid-one epoxide accumulation could be increased up to a 100 fold within 72 h by elicitation through addition of a suspension of either living free or immobilized yeasts, of dead RHODOTORULA RUBRA cells or crude cell wall fraction. Chitosan and alginate, exhibiting elicitor properties in other systems, scarcely had an effect, arachidonic acid and elaidic acid were ineffective. The elicitors of acridone epoxide accumulation did not effect an increase of rutacridone in RUTA cultures.

Purification and specificity of a human microsomal epoxide hydratase.

Epoxide hydratase was solubilized from human liver microsomal fractions and purified to an extent where the specific activity was 40-fold greater than that of the liver homogenate. Combination of homogenate and purified preparation showed that the increase in activity was not due to the removal of an inhibitor. Monosubstituted oxiranes with a lipophilic substituent larger than an ethyl group (isopropyl, t-butyl, n-hexyl, phenyl) readily interacted as substrates or inhibitors with this purified human epoxide hydratase, whereas those with a small substituent (methyl, ethyl, vinyl) were inactive, probably reflecting greater affinity of the former epoxides owing to lipophilic binding sites near the active site of the enzyme. In a series of oxiranes having a lipophilic substituent of sufficient size (styrene oxides), monosubstituted as well as 1,1- and cis-1,2-disubstituted oxiranes readily served as substrates or inhibitors of the enzyme, but not the trans-1,2-disubstituted, tri- or tetra-substituted oxiranes. trans-Substitution at the oxirane ring apparently prevents access of the oxirane ring to the active site by steric hindrance. Epoxide hydratase was also solubilized from microsomal fractions of rat and guinea-pig liver and purified by the same procedure. Structural requirements for effective interaction of substrates, inhibitors and activators were qualitatively identical for epoxide hydratase from the three sources. However, several quantitative differences were observed. Thus human hepatic epoxide hydratase seems to be very similar to, although not identical with, the enzyme from guinea pig or rat. Studies with epoxide hydratase from the latter two species therefore appear to be significant with respect to man. In addition, knowledge of structural requirements for epoxides to serve as substrates for human epoxide hydratase may prove useful for drug design. Compounds which need aromatic or olefinic moieties for their desired effect would not be expected to lead to accumulation of epoxides if their structure was such as to allow for a metabolically produced epoxide to be rapidly consumed by epoxide hydratase.