Candida tropicalis Etr1p and Saccharomyces cerevisiae Ybr026p (Mrf1'p), 2-enoyl thioester reductases essential for mitochondrial respiratory competence

We report here on the identification and characterization of novel 2-enoyl thioester reductases of fatty acid metabolism, Etr1p from Candida tropicalis and its homolog Ybr026p (Mrf1'p) from Saccharomyces cerevisiae. Overexpression of these proteins in S. cerevisiae led to the development of significantly enlarged mitochondria, whereas deletion of the S. cerevisiae YBR026c gene resulted in rudimentary mitochondria with decreased contents of cytochromes and a respiration-deficient phenotype. Immunolocalization and in vivo targeting experiments showed these proteins to be predominantly mitochondrial. Mitochondrial targeting was essential for complementation of the mutant phenotype, since targeting of the reductases to other subcellular locations failed to reestablish respiratory growth. The mutant phenotype was also complemented by a mitochondrially targeted FabI protein from Escherichia coli. FabI represents a nonhomologous 2-enoyl-acyl carrier protein reductase that participates in the last step of the type II fatty acid synthesis. This indicated that 2-enoyl thioester reductase activity was critical for the mitochondrial function. We conclude that Etr1p and Ybr026p are novel 2-enoyl thioester reductases required for respiration and the maintenance of the mitochondrial compartment, putatively acting in mitochondrial synthesis of fatty acids.     

Peroxisomal Delta(3),Delta(2)-enoyl CoA isomerases and evolution of cytosolic paralogues in embryophytes

Delta(3),Delta(2)-enoyl CoA isomerase (ECI) is an enzyme that participates in the degradation of unsaturated fatty acids through the beta-oxidation cycle. Three genes encoding Delta(3),Delta(2)-enoyl CoA isomerases and named AtECI1, AtECI2 and AtECI3 have been identified in Arabidopsis thaliana. When expressed heterologously in Saccharomyces cerevisiae, all three ECI proteins were targeted to the peroxisomes and enabled the yeast Deltaeci1 mutant to degrade 10Z-heptadecenoic acid, demonstrating Delta(3),Delta(2)-enoyl CoA isomerase activity in vivo. Fusion proteins between yellow fluorescent protein and AtECI1 or AtECI2 were targeted to the peroxisomes in onion epidermal cells and Arabidopsis root cells, but a similar fusion protein with AtECI3 remained in the cytosol for both tissues. AtECI3 targeting to peroxisomes in S. cerevisiae was dependent on yeast PEX5, while expression of Arabidopsis PEX5 in yeast failed to target AtECI3 to peroxisomes. AtECI2 and AtECI3 are tandem duplicated genes and show a high level of amino acid conservation, except at the C-terminus; AtECI2 ends with the well conserved peroxisome targeting signal 1 (PTS1) terminal tripeptide PKL, while AtECI3 possesses a divergent HNL terminal tripeptide. Evolutionary analysis of ECI genes in plants revealed several independent duplication events, with duplications occurring in rice and Medicago truncatula, generating homologues with divergent C-termini and no recognizable PTS1. All plant ECI genes analyzed, including AtECI3, are under negative purifying selection, implying functionality of the cytosolic AtECI3. Analysis of the mammalian and fungal genomes failed to identify cytosolic variants of the Delta(3),Delta(2)-enoyl CoA isomerase, indicating that evolution of cytosolic Delta(3),Delta(2)-enoyl CoA isomerases is restricted to the plant kingdom.     

Characterization of 2-enoyl thioester reductase from mammals. An ortholog of YBR026p/MRF1'p of the yeast mitochondrial fatty acid synthesis type II

A data base search with YBR026c/MRF1', which encodes trans-2-enoyl thioester reductase of the intramitochondrial fatty acid synthesis (FAS) type II in yeast (Torkko, J. M., Koivuranta, K. T., Miinalainen, I. J., Yagi, A. I., Schmitz, W., Kastaniotis, A. J., Airenne, T. T., Gurvitz, A., and Hiltunen, K. J. (2001) Mol. Cell. Biol. 21, 6243-6253), revealed the clone AA393871 (HsNrbf-1, nuclear receptor binding factor 1) in human EST data bank. Expression of HsNrbf-1, tagged C-terminally with green fluorescent protein, in HeLa cells, resulted in a punctated fluorescence signal, superimposable with the MitoTracker Red dye. Wild-type polypeptide was immunoisolated from the extract of bovine heart mitochondria. Recombinant HsNrbf-1p reduces trans-2-enoyl-CoA to acyl-CoA with chain length from C6 to C16 in an NADPH-dependent manner with preference to medium chain length substrate. Furthermore, expression of HsNRBF-1 in the ybr026cDelta yeast strain restored mitochondrial respiratory function allowing growth on glycerol. These findings provide evidence that Nrbf-1ps act as a mitochondrial 2-enoyl thioester reductase, and mammalian cells may possess bacterial type fatty acid synthetase (FAS type II) in mitochondria, in addition to FAS type I in the cytoplasm.     

Induction of rat liver mitochondrial fatty acid elongation by the administration of peroxisome proliferator di-(2-ethylhexyl)phthalate: absence of elongation activity in peroxisomes

The administration of di-(2-ethylhexyl)phthalate (DEHP)3 to male Sprague-Dawley rats resulted in more than a threefold increase in activity of acetyl CoA-dependent hepatic mitochondrial fatty acid elongation. Peroxisomes obtained either from control or DEHP-treated rats were not capable of elongating any of the fatty acyl CoAs tested. Furthermore, the peroxisomes possessed no trans-2-enoyl CoA reductase activity. Therefore, the elongation activity in the 7500g fraction from both control and DEHP-fed animals can be attributed totally to the mitochondria. Maximal incorporation of acetyl CoA occurred in the presence of both NADH and NADPH, and octanoyl CoA (8:0) and decanoyl CoA (10:0) were found to be optimal primers for fatty acid elongation in both control and DEHP-treated animals. The apparent Km for 8:0 CoA was 17 microM in both animal groups while the Vmax was increased from 4.5 to 12.5 nmol/min/mg following treatment. The apparent Km for 10:0 CoA was 10 microM in both control and DEHP-treated groups while the apparent Vmax increased from 2.5 to 10 nmol/min/mg; palmitoyl-CoA (16:0) was a very poor primer for chain elongation. Although the acetyl CoA-dependent fatty acid elongation was stimulated by DEHP treatment, the mitochondrial trans-2-enoyl CoA reductase activity was unaffected. The mitochondrial total elongation activity following DEHP-treatment using 8:0 CoA as primer was about two times higher than enoyl CoA reductase activity using trans-2-decenoyl CoA (10:1). This was the result of accumulation of intermediates, which were identified as trans-2-10:1 (35%), beta-hydroxy 10:0 (25%), unidentified (15%), and elongated saturated product 10:0 (24%). Elongation by one acetate unit was found in both the control and DEHP-treated animals. The results are discussed in terms of physiological significance.     

Equilibrium sorption isotherm for metal ions on tree fern

A new sorbent system for removing heavy metal ions, such as Zn(II), Cu(II) and Pb(II), from aqueous solutions has been investigated. This new sorbent is tree fern, an agriculture product. Variables of the system include solution temperature and sorbent particle size. The experimental results were fitted to the Langmuir, Freundlich and Redlich–Peterson isotherms to obtain the characteristic parameters of each model. Both the Langmuir and Redlich–Peterson isotherms were found to well represent the measured sorption data. According to the evaluation using the Langmuir equation, the maximum sorption capacities of metal ions onto tree fern were 7.58 mg/g for Zn(II), 10.6 mg/g for Cu(II) and 39.8 mg/g for Pb(II). It was noted that an increase in temperature resulted in a higher metal loading per unit weight of the sorbent. Decreasing the particle sizes of tree fern led in an increase in the metal uptake per unit weight of the sorbent.     

Kinetics of biosorption of cadmium on Baker's yeast

In the present study the kinetics of biosorption of cadmium(II) ions by deactivated protonated yeast converted to sodium form was investigated for different initial concentrations of the metal ion (10-100 ppm) and different sorbent dosages (0.1-2.0 g) at a pH of 6.5. The adsorption process occurred in four distinct steps and the rates for these steps decreased sequentially. The rate of cadmium uptake in each case was pseudo-second-order with respect to metal ion concentration. The amount sorbed at equilibrium was found to be directly proportional to the initial metal ion concentration divided by the sorbent mass.     

Dual action of 2-decynoyl coenzyme A: inhibitor of hepatic mitochondrial trans-2-enoyl coenzyme A reductase and peroxisomal bifunctional protein and substrate for the mitochondrial beta-oxidation system

The present study was designed to determine the action of the 2-acetylenic acid thioester on mitochondrial fatty acid chain elongation and beta-oxidation. Addition of 2-decynoyl CoA to a rat liver mitochondrial suspension resulted in a significant stimulation of the rate of oxidation of NADPH and NADH. This enhanced oxidation rate was not due to the mitochondrial trans-2-enoyl CoA reductase-catalyzed conversion of the 2-acetylenic acid thioester to the saturated product, decanoate, as measured by gas-liquid chromatography. On the contrary, the mitochondrial trans-2-enoyl CoA reductase activity was markedly inhibited by the 2-acetylenic acid derivative, as evidenced by the decrease in the reduction of trans-2-decenoyl CoA to decanoic acid. Incubation of the mitochondrial fraction with either NADPH or NADH and 2-decynol CoA resulted in the gas chromatographic identification of three products: beta-ketodecanoate, beta-hydroxydecanoate, and trans-2-decenoate. In the absence of reduced pyridine nucleotide, a single product was formed and identified as beta-ketodecanoate. Confirmation of the identity of this product was obtained by the observation of the formation of the Mg2+-enolate complex (303-nm absorbance peak). These results suggest that, although the 2-decynoyl CoA is an inhibitor of mitochondrial trans-2-enoyl CoA reductase activity, it is a substrate for the mitochondrial trans-2-enoyl CoA hydratase (crotonase). This was confirmed by incubation of 2-decynoyl CoA with commercially purified liver mitochondrial crotonase. The beta-ketodecanoate is formed in a two-step process: hydration of the 2-decynoyl CoA to an unstable enol intermediate which undergoes rearrangement to the beta-ketodecanoyl CoA. Interestingly, although the mitochondrial crotonase can utilize the 2-acetylenic acid thioesters, this was not the case for the peroxisomal bifunctional hydratase which was markedly inhibited by varying concentrations of 2-decynoyl CoA.     

Novel metabolite structures from biotransformation of a sesquiterpenoid ketone by selected fungal strains

The sesquiterpenoid ketone, 1,4,4-trimethyltricyclo[5.4.0.0(3.5)]undec-7-en-9-one (1), was subjected to microbial transformation by six fungal strains: Aspergillus niger ATCC 9142, Aspergillus ochraceus DSM 824, Beauveria bassiana ATCC 7159, Cunninghamella echinulata ATCC 9244, Rhizopus arrhizus ATCC 11.145, and Absidia blakesleeana ATCC 10.148. Four main metabolites were formed from 1: 10(R)- and 10(S)-hydroxy-1,4,4-trimethyltricyclo-[5.4.0.0(3.5)]undec-7- en- 9-one (2 and 3, respectively), besides 4(R)- and 4(S)-(hydroxymethyl)-1,4-dimethyltricyclo[5.4.0.0(3.5)]undec -7-en-9-one (4 and 5, respectively). Compounds 2-5 were isolated with varying percentages from the respective transformations, and their structures established unequivocally by a combination of spectroscopic methods. Metabolites 2 and 3 are products of hydroxylation at C-10, in either R- or S-position; in 4 and 5, one geminal CH3 group each on the cyclopropane ring has been transformed into a CH2OH function.     

Myocardial Overexpression of Mecr,a Gene of Mitochondrial FAS II Leads to Cardiac Dysfunction in Mouse

It has been recently recognized that mammalian mitochondria contain most, if not all, of the components of fatty acid synthesis type II (FAS II). Among the components identified is 2-enoyl thioester reductase/mitochondrial enoyl-CoA reductase (Etr1/Mecr), which catalyzes the NADPH-dependent reduction of trans-2-enoyl thioesters, generating saturated acyl-groups. Although the FAS type II pathway is highly conserved, its physiological role in fatty acid synthesis, which apparently occurs simultaneously with breakdown of fatty acids in the same subcellular compartment in mammals, has remained an enigma. To study the in vivo function of the mitochondrial FAS in mammals, with special reference to Mecr, we generated mice overexpressing Mecr under control of the mouse metallothionein-1 promoter. These Mecr transgenic mice developed cardiac abnormalities as demonstrated by echocardiography in vivo, heart perfusion ex vivo, and electron microscopy in situ. Moreover, the Mecr transgenic mice showed decreased performance in endurance exercise testing. Our results showed a ventricular dilatation behind impaired heart function upon Mecr overexpression, concurrent with appearance of dysmorphic mitochondria. Furthermore, the data suggested that inappropriate expression of genes of FAS II can result in the development of hereditary cardiomyopathy.     

Acylated protopanaxadiol-type ginsenosides from the root of Panax ginseng

Six new protopanaxadiol-type ginsenosides, named ginsenosides Ra(4) -Ra(9) (1-6, resp.), along with 14 known dammarane-type triterpene saponins, were isolated from the root of Panax ginseng, one of the most important Chinese medicinal herbs. The structures of the new compounds were determined by spectroscopic methods, including 1D- and 2D-NMR, HR-MS, and chemical transformation as (20S)- 3-O-{β-D-6-O-[(E)-but-2-enoyl]glucopyranosyl-(1→2)-β-D-glucopyranosyl}-20-O-[β-D-xylopyranosyl-(1→4)-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (1), (20S)-3-O-[β-D-6-O-acetylglucopyranosyl-(1→2)-β-D-glucopyranosyl]-20-O-[β-D-xylopyranosyl-(1→4)-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (2), (20S)-3-O-{β-D-6-O-[(E)-but-2-enoyl]glucopyranosyl-(1→2)-β-D-glucopyranosyl}-20-O-[β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (3), (20S)-3-O-{β-D-6-O-[(E)-but-2-enoyl]glucopyranosyl-(1→2)-β-D-glucopyranosyl}-20-O-[α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (4), (20S)-3-O-{β-D-4-O-[(E)-but-2-enoyl]glucopyranosyl-(1→2)-β-D-glucopyranosyl}-20-O-[α-L-arabinofuranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (5), (20S)-3-O-{β-D-6-O-[(E)-but-2-enoyl]glucopyranosyl-(1→2)-β-D-glucopyranosyl}-20-O-[α-L-arabinofuranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (6). The sugar moiety at C(3) of the aglycone of each new ginsenoside is butenoylated or acetylated.     

Ternary biosorption studies of Cd(II), Cr(III) and Ni(II) on shelled Moringa oleifera seeds

Competitive biosorption of Cd(II), Cr(III) and Ni(II) on unmodified shelled Moringa oleifera seeds (SMOS) present in ternary mixture were compared with the single metal solution. The extent of adsorption capacity of the ternary metal ions tested on unmodified SMOS was low (10-20%) as compared to single metal ions. SMOS removed the target metal ions in the selectivity order of Cd(II) > Cr(III) > Ni(II). Sorption equilibria, calculated from adsorption data, explained favorable performance of biosorption system. Regeneration of exhausted biomass was also attempted for several cycles with a view to restore the sorbent to its original state.     

Use of monoclonal antibodies for purifying the regulatory subunit of cAMP-dependent protein kinase

Monoclonal antibodies against regulatory subunit of cAMP-dependent protein kinase, type II, were obtained from pig brain (R II). The immune-affinity sorbent has been synthesized on the basis of monoclonal antibodies against R II. The method was proposed for the purification of homogeneous R II with high cAMP-binding activity using immune-affinity sorbent.     

Sesquiterpenes from the wood of Juniperus lucayana

Bioassay-guided fractionation of ethanolic extract from the wood of Juniperus lucayana afforded three sesquiterpenes named 3-hydroxypseudowiddran-6(7)-en-4-ol (1), 15-hydroxyallo-cedrol (2) and 12-hydroxywiddrol (3) together with six known sesquiterpenes (4-9) and two known flavonoids (10 and 11). Their structures were established on the basis of comprehensive spectroscopic analyses, including 2D NMR spectroscopy and mass spectrometry. The structures of compounds were identified as 1alpha,4beta,11alpha,11beta-tetramethylbicyclo[5,4,0]undec-6(7)-en-3alpha, 4alpha-diol (1), 4beta-hydroxymethyl-5,5,9beta-trimethyltricyclo[4.3.0.2(1.4)]undecan-3alpha-ol (2) and 4beta-hydroxymethyl-7alpha,11alpha,11beta-trimethylbicyclo [5.4.0]undec-1-en-4alpha-ol (3). The major compounds isolated were evaluated for their antifungal activity against Botrytis cinerea. Widdrol (7) was the most active, reaching the 71% inhibition level on mycelial growth after 6 days.     

Do rat hepatic microsomes contain multiple NADPH-supported fatty acid chain elongation pathways or a single pathway?

[2-14C]-trans-2-hexadecenoyl CoA (16:1) and [2-14C]-trans-2-cis-8,11,14-eicosatetraenoyl CoA (20:4) were chemically synthesized and employed as competitive substrates for the liver microsomal trans-2-enoyl CoA reductase component of the fatty acid chain elongation system. Both 7.5 microM and 15 microM 20:4 competitively inhibited the reduction of 16:1 CoA to palmitoyl CoA. In addition, the reduction of both substrates was identically inhibited to the same extent by the acetylenic derivative, dec-2-ynoyl CoA. Furthermore, trypsin, chymotrypsin and subtilisin inhibited trans-2-enoyl CoA reductase activity when three different substrates were employed--16:1, 20:4 and trans-2-cis-11-octadecadienoyl CoA (18:2). These results are consistent with the hypothesis of multiple condensing enzymes connected to a single elongation pathway.     

Cytotoxic clerodane diterpenes from Glossocarya calcicola

Three clerodane diterpenes were isolated and identified from leaf extract of Glossocarya calcicola. Compound has been characterised as (rel)-10betaH-trans-12xi-(2-methylbut-2(E)-enoyl)-1beta-(isobutanoyl)-6alpha,13xi-dihydroxyclerodan-4(20),8(18)-dien-7,15-dione-15,16-oxide, to which we have assigned the trivial name calcicolin-A. The other two compounds had the same skeletal structure and C-12 substituent but in compound, the C-1 esterifying group becomes 2-methylbut-2(E)-enoic acid and in it becomes 2-methylbutanoic acid. Although anti-insect activity was not observed for G. calcicola, cytotoxicity against insect and human carcinoma cell lines was detected.     

Hg(II) adsorption by <Emphasis Type="Italic">Bacillus mucilaginosus</Emphasis>: mechanism and equilibrium parameters

Our previous findings have indicated that Bacillus mucilaginosus might be a promising biosorbent. However, up to now, few studies have been performed to examine the use of B. mucilaginosus as a sorbent, especially as a sorbent for Hg(II). The aim of the current study was to investigate the adsorption of Hg(II) by B. mucilaginosus and the underlying mechanism involved. The results showed that B. mucilaginosus exhibited effective adsorption of Hg(II), and the experimental data were well fitted by the Langmuir model with equilibrium constant of 3.32 × 10⁴ M⁻¹ and maximum adsorption capacity of 393 mg(Hg)/l(bacterial culture). The average saturated adsorption amount of Hg(II) by each cell was 9.83 × 10⁹ atoms, with time to reach adsorption equilibrium less than 10 min. The adsorption efficiency was mainly dependent on pH. Surface adsorption of capsules was identified to be the major mechanism for the biosorption of Hg(II) by B. mucilaginosus, which might be associated with the cell products on the surface of capsules of B. mucilaginosus. Differences observed in adsorption behaviors at different concentrations of Hg(II) were well explained using the Visual minTEQ software. Our findings might shed some lights on the application of B. mucilaginosus as an adsorbent for Hg(II) and other heavy metals.     

A Root Growth-promoting Factor,Capillarol, from Artemisia capillaris Thunb

A root growth-promoting factor was isolated from Artemisia capillaris Thunb. The chemical structure of this compound was elucidated as methyl 3-(3-methylbut-2-enoyl)-4-hydroxycinnamate (capillarol) on the basis of its spectroscopic analysis. At 5×10^–4m this compound promoted rice root growth to 180% of the control value.     

Features of protein adsorption by silicopolymethylsiloxanes

The adsorption of tripsin and albumin on silicapolymetylsilocsan (SG-PMS) and its modified forms by cooper (II)--(SG-PMS (Cu)--from water-salt solutions were studied. It was determined difference in peculiarities of proteins sorption depending on its amino acids composition, chemistry of sorbent surface and acidity of medium. It was showed, that modified by cooper (II) sorbent have high affinity to tripsin than albumin in studied solutions. Influence of bearer modification degree on albumin immobilisation and interaction character of SG-PMS (Cu) active centres with function groups of enzyme and albumin have been showed.     

Possible role of NADPH-dependent enoyl coenzyme A reductase in -oxidation of unsaturated fatty acids

An 2-enoyl CoA reductase from rat liver mitochondria catalyzes the reduction of both oct-$\text{cis}$-2-enoyl CoA and its $\text{trans}$ isomer in the presence of NADPH as a specific electron donor. This reductase is solubilized from mitochondria by sonication. The possible role of the reductase in the β-oxidation pathway of the unsaturated fatty acids is discussed.