2(S),4(R)-4-(β-d-Galactopyranosyloxy)-4-isobutyl-glutamic acid: A new amino acid in Reseda odorata

2(S),4(R)-4-(β-d-Galactopyranosyloxy)-4-isobutylglutamic acid (I) has been isolated from the flowers of Reseda odorata, wherein it occurs in substantial quantity. Hydrolysis of I gives d-galactose, 2(S),4(R)-4-hydroxy-4-isobutylglutamic acid (II) and 3(R),5(S)-3-hydroxy-3-isobutyl-2-pyrrolidone-5-carboxylic acid (III) and its treatment with nitrous acid yields a galactoside of a non-nitrogenous hydroxy acid lactone (IV). The structures of I and its degradation products are supported by PMR, ¹³C-NMR and other spectroscopic methods. ¹³C-NMR spectroscopy of the model compound 2-(β-d-galactopyranosyloxy)isobutyric acid confirmed the structure of the natural product. The S- (or l-) configuration at C(2) in the amino acid moiety of I has been established by the use of the Clough—Lutz—Jirgenson rule and the R-configuration at C(4) of the same unit has been assigned tentatively. I represents the first example of a glycoside of a higher plant amino acid in which the carbohydrate residue is linked to an aliphatic hydroxy group.     

Regulation of mitochondrial biogenesis: yeast mutants deficient in synthesis of delta-aminolevulinic acid

A new class of Saccharomyces cerevisiae mutants deficient in biosynthesis of all cytochromes was isolated from cultures grown in medium containing ethidium bromide. Cytochrome c synthesis may be restored to normal by growing mutant cells in medium supplemented with δ-aminolevulinic acid. Cytochrome deficiency results from mutation in two genetic determinants, one nuclear, the other mitochondrial. When cells possess normal (ρ⁺) mitochondrial DNA, expression of the abnormal nuclear determinant (cyd-1) is largely masked, so that cells can grow on glycerol as primary carbon source and all cytochromes are present. Nevertheless, the presence of the cyd-1 mutation may be detected in ρ⁺ strains, since synthesis of all cytochromes is enhanced to some extent by δ-aminolevulinic acid. Destruction of mitochondrial DNA unmasks the underlying defect so that cyd-1 ρ^? strains are almost completely lacking in detectable cytochromes. Although spectra of cyd-1 ρ⁺ strains resemble those of cytochrome c (cyc) mutants, cyd-1 mutants represent a new complementation group different from six known cyc groups. Cytochrome c biosynthesis in only one of these six types of cytochrome c mutants, cyc4-1, was restored to normal by δ-aminolevulinic acid. Therefore, since cyc4-1 and cyd-1 are complementary, and segregate independently, δ-aminolevulinic acid synthesis appears to be controlled by at least two nuclear genes, and by one or more genes located in mitochondrial DNA. Glycine does not replace δ-aminolevulinic acid in stimulating cytochrome biosynthesis in cyd-1 or cyc-4 mutants. A regulatory system involving exchange of information between mitochondria and the nuclear-cytosolic compartment is indicated by the results. Studies with isolated mitochondria indicate that a limitation of intra-cellular δ-aminolevulinic acid supply is reflected in mitochondrial composition, not just in numbers of organelles.     

Adaptive evolution improves acid tolerance and succinic acid production in Actinobacillus succinogenes

Fermentation at low pH is an efficient way to improve the competitiveness of biological succinic acid-producing process. Actinobacillus succinogenes shows good performance of succinic acid production under anaerobic conditions, but its succinic acid production capability at the low-pH is inefficient due to the poor acid resistance. Herein, a mutant A. succinogenes BC-4 with improved cell growth and succinic acid production under weak acid conditions was obtained by adaptive evolution. The specific growth rate and succinic acid production of BC-4 reached 0.13 g/L/h and 20.77 g/L, which were increased by 3.25- and 2.95- fold, respectively compared with the parent strain under anaerobic condition at pH 5.8. The activities of specific enzymes with ATP generation were significantly enhanced under weak acidic conditions, resulting in 1.28-fold increase in the maximum ATP level. Membrane fatty acid composition analysis demonstrated that the ratio of saturated to unsaturated fatty acids was decreased from 1.62 to 1.44 in mutant BC-4, leading to improved intracellular pH homeostasis. Furthermore, the change from long-chain to median-chain fatty acid might lower the permeability of H⁺ into cytoplasm for survival under acid stress. These results indicated that A. succinogenes BC-4 is a promising candidate for succinic acid production under weak acid condition.     

Absolute configuration of ceriporic acids, the iron redox-silencing metabolites produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora

Ceriporic acids are a class of alk(en)ylitaconic acids produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora. The unique function of alkylitaconic acid is the redox silencing of the Fenton reaction system by inhibiting reduction of Fe³⁺. Ceriporic acids have an asymmetric centre at carbon-3, but absolute configuration has not been determined. We have isolated a series of ceriporic acids from the cultures of C. subvermispora, and measured their NMR spectra using a chiral shift reagent. In comparison with NMR spectra of (R)-(−)- and (S)-(+)-methylsuccinic acid and those of natural and chemically synthesized racemic mixtures of ceriporic acids, we have determined the absolute configuration of ceriporic acids as (R)-3-tetradecylitaconic acid (ceriporic acid A), (R)-3-hexadecylitaconic acid (ceriporic acid B) and (R,Z)-2-(hexadec-7-enyl)-3-itaconic acid (ceriporic acid C). We herein discuss their stereoselective biosynthetic pathway and the structural diversity of fungal secondary metabolites.     

An active site mutant of Escherichia coli cyclopropane fatty acid synthase forms new non-natural fatty acids providing insights on the mechanism of the enzymatic reaction

We have produced and purified an active site mutant of the Escherichia coli cyclopropane fatty acid synthase (CFAS) by replacing the strictly conserved G236 within cyclopropane synthases, by a glutamate residue, which corresponds to E146 of the homologous mycolic acid methyltransferase, Hma, producing hydroxymethyl mycolic acids. The G236E CFAS mutant had less than 1% of the in vitro activity of the wild type enzyme. We expressed the G236E CFAS mutant in an E. coli (DE3) strain in which the chromosomal cfa gene had been deleted. After extraction of phospholipids and conversion into the corresponding fatty acid methyl esters (FAMEs), we observed the formation of cyclopropanated FAMEs suggesting that the mutant retained some of the normal activity in vivo. However, we also observed the formation of new C17 methyl-branched unsaturated FAMEs whose structures were determined using GC/MS and NMR analyses. The double bond was located at different positions 8, 9 or 10, and the methyl group at position 10 or 9. Thus, this new FAMEs are likely arising from a 16:1 acyl chain of a phospholipid that had been transformed by the G236E CFAS mutant in vivo. The reaction catalyzed by this G236E CFAS mutant thus starts by the methylation of the unsaturated acyl chain at position 10 or 9 yielding a carbocation at position 9 or 10 respectively. It follows then two competing steps, a normal cyclopropanation or hydride shift/elimination events giving different combinations of alkenes. This study not only provides further evidence that cyclopropane synthases (CSs) form a carbocationic intermediate but also opens the way to CSs engineering for the synthesis of non-natural fatty acids.     

Characterization of cis-9 trans-11 trans-15 C18:3 in milk fat by GC and covalent adduct chemical ionization tandem MS

Rumen biohydrogenation of dietary α-linolenic acid gives rise in ruminants to accumulation of fatty acid intermediates, some of which may be transferred into milk. Rumelenic acid [cis-9 trans-11 cis-15 C18:3 (RLnA)] has recently been characterized, but other C18:3 minor isomers are still unknown. The objective of this work was to identify a new isomer of octatridecenoic acid present in milk fat from ewes fed different sources of α-linolenic acid. Structural characterization of this fatty acid was achieved by GC-MS. Analysis of dimethyloxazoline and picolinyl ester derivatives allowed for location of the double bond positions. Covalent adduct chemical ionization tandem mass spectrometry confirmed the positional structure 9-11-15, identical to RLnA, and helped to establish double bond geometry (cis-trans-trans). This new C18:3 isomer could be formed by isomerization of cis-15 bond of RLnA and subsequently converted by hydrogenation to trans-11 trans-15 C18:2, an octadecadienoic acid also detected in this study.     

Acid-catalyzed transformation of 13(S)-hydroperoxylinoleic acid into epoxyhydroxyoctadecenoic and trihydroxyoctadecenoic acids

Acid treatment of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid in tetrahydrofuran-water solvent afforded mainly (11R,12R,13S)-(Z)-12,13-epoxy-11-hydroxy-9-octadecenoic acid, diastereomeric (Z)-11,12,13-trihydroxy-9-octadecenoic acids and four isomers of (E)-9,12,13(9,10,13)-trihydroxy-10(11)-octadecenoic acid. Other minor products were oxooctadecadienoic, (E)-9(13)-hydroxy-13(9)-oxo-10(11)-octadecenoic and (E)-12-oxo-10-dodecenoic acids. A heterolytic mechanism for acid catalysis was indicated, even though most of the products characterized also have been observed as a result of homolytic decomposition of the hydroperoxide via an oxy radical. Most of the products found in this study have been observed as metabolites of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadenoic acid in biological systems, and analogous compounds have been reported as metabolites of (12S)-(5Z,8Z,10E, 14Z)-12-hydroperoxy-5,8,10,14-hydroperoxy-5,8,10,14-eicosatetraenoic acid in either blood platelets or lung tissue.     

Partial purification and characterization of the lipoxygenase from grains of Hordeum distichum

Lipoxygenase activities in ungerminated and germinating barley grains were found to be associated exclusively with the embryos. A lipoxygenase was extracted from ungerminated embryos and partially purified by fractional precipitation with ammonium sulfate and gel-filtration. Both the crude extracts and the purified preparation appeared to contain only a fatty acid type lipoxygenase which mainly converted linolele acid to 9-hydroperoxy, trans-10, cis-12-octadecadienoic acid. The purified enzyme was inhibited by its own products, hydroperoxides, but not by 1 mM cyanide, EDTA, Hg²⁺ or Cu²⁺.     

Conversion of (2Z,4E)-5-(1′,2′-epoxy-2′,6′,6′-trimethylcyclohexyl)-3-methyl-2,4-pentadienoic acid to xanthoxin acid by Cercospora cruenta, a fungus producing (+)-abscisic acid

(±)-(2Z,4E)-5-(1′,2′-epoxy-2′,6′,6′-trimethylcyclohexyl)-3-methyl-2,4-pentadienoic acid was metabolized by Cercospora cruenta, which has the ability to produce (+)-abscisic acid (ABA), to give (±)-(2Z,4E)-xanthoxin acid, (±)-(2Z,4E)-5′-hydroxy-1′,2′-epoxy-1′,2′-dihydro-β-ionylideneacetic acid, (±)-1′,2′-epoxy-1′,2′-dihydro-β-ionone and trace amounts of ABA.     

Influence of lignin-derived phenolic compounds on the Clostridium thermocellum endo-β-1,4-xylanase XynA

Phenolic compounds released during pretreatment of lignocellulosic biomass influence its enzymatic hydrolysis. To understand the effects of these compounds on the kinetic properties of xylan-degrading enzymes, the present study employed the recombinant cellulosomal endo-β-1,4-xylanase, thermostable GH11 XynA protein from Clostridium thermocellum, as an enzyme model to evaluate the effects of 4-hydroxybenzoic acid, gallic acid, vanillin, tannic acid, p-coumaric acid, ferulic acid, syringaldehyde, and cinnamic acid. XynA was deactivated by the assayed phenols at 60 °C, presenting the strongest deactivation in the presence of tannic acid, with an activity reduction of about 80 %. Thermal stability of XynA was influenced by ferulic acid, syringaldehyde, cinnamic acid, 4-hydroxybenzoic acid, and p-coumaric acid. The hydrolysis rate of oat-spelt xylan by XynA was influenced by temperature, being unable to hydrolyze at 40 °C in the presence of tannic acid. On hydrolysis at 60 °C, the presence of gallic and tannic acid caused a major reduction in reducing sugar production, generating 3.74 and 2.15 g.L^-1 of reducing sugar, respectively, whereas the reaction in the absence of phenols generated 4.41 g.L^-1. When XynA was pre-deactivated by phenols it could recover most of its activity at 40 °C, however, at 60 °C activity could not be reestablished.     

Formation of (-⊃-1′,2′-epi-2-cis-xanthoxin acid from a precursor of abscisic acid

Tomato shoots and avocado mesocarp supplied with (±)-[2-¹⁴C]-5-(1,2-epoxy-2,6,6-trimethylcyclohexyl)-3-methylpenta-cis-2-trans-4-dienoic acid metabolize it into (+)-abscisic acid and a more polar material that was isolated and identified as (?)-epi-1′(R),2′(R)-4′(S)-2-cis-xanthoxin acid. The (+)-1′(S),2′(S)-4′(S)-2-cis-xanthoxin acid recently synthesized from natural violaxanthin, has the 1′,2′-epoxy group on the opposite side of the ring to that of the 4′(S)-hydroxyl group and the compound is rapidly converted into (+)-abscisic acid. The 1′,2′-epoxy group of (?)-1′,2′-epi-2-cis-xanthoxin acid is on the same side of the ring as the 4′(S) hydroxyl group: the compound is not metabolized into abscisic acid. The configuration of the 1′,2′-epoxy group probably controls whether or not the 4′(S) hydroxyl group can be oxidized. (+)-2-cis-Xanthoxin acid is probably not a naturally occurring intermediate because a ‘cold trap’, added to avocado fruit forming [¹⁴C]-labelled abscisic acid from [2-¹⁴C]mevalonate, failed to retain [¹⁴C] label.     

Synthesis and biological activity of hydroxylated derivatives of linoleic acid and conjugated linoleic acids

Allylic hydroxylated derivatives of the C18 unsaturated fatty acids were prepared from linoleic acid (LA) and conjugated linoleic acids (CLAs). The reaction of LA methyl ester with selenium dioxide (SeO₂) gave mono-hydroxylated derivatives, 13-hydroxy-9Z,11E-octadecadienoic acid, 13-hydroxy-9E,11E-octadecadienoic acid, 9-hydroxy-10E,12Z-octadecadienoic acid and 9-hydroxy-10E,12E-octadecadienoic acid methyl esters. In contrast, the reaction of CLA methyl ester with SeO₂ gave di-hydroxylated derivatives as novel products including, erythro-12,13-dihydroxy-10E-octadecenoic acid, erythro-11,12-dihydroxy-9E-octadecenoic acid, erythro-10,11-dihydroxy-12E-octadecenoic acid and erythro-9,10-dihydroxy-11E-octadecenoic acid methyl esters. These products were purified by normal-phase short column vacuum chromatography followed by high-performance liquid chromatography (HPLC). Their chemical structures were characterized by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR). The allylic hydroxylated derivatives of LA and CLA exhibited moderate in vitro cytotoxicity against a panel of human cancer cell lines including chronic myelogenous leukemia K562, myeloma RPMI8226, hepatocellular carcinoma HepG2 and breast adenocarcinoma MCF-7 cells (IC₅₀ 10-75 μM). The allylic hydroxylated derivatives of LA and CLA also showed toxicity to brine shrimp with LD₅₀ values in the range of 2.30-13.8 μM. However these compounds showed insignificant toxicity to honeybee at doses up to 100 μg/bee.     

GC-MS identification of abscisic acid and abscisic acid metabolites in seed of Vigna unguiculata

Abscisic acid, phaseic acid and 4′-dihydrophaseic acid were identified by GC-MS of derivatized (Me, MeTMSi) extracts from immature fruits of Vigna unguiculata. The fruits also contained some other ABA-related compounds, one of which might be epi-4′-dihydrophaseic acid while another was tentatively identified as 6′-hydroxymethylabscisic acid.     

Diterpene acids from Helianthus species and their microbiological conversion by Gibberella fujikuroi, mutant B1-41a

The diterpene acid content in 10 species of Helianthus has been investigated. Ent-12,16-cyclokauranoic acid, isolated from H. annuus, is converted into a series of 12,16-cyclogibberellins by cultures of Gibberella fujikuroi, mutant B1-41a, and 12,16-cyclogibberellins A₉, and A₁₂ have been isolated. Ent-12β-acetoxykaurenoic acid and ent-13(S)-angeloxyatisenoic acid have been isolated from H. decapetalus; the metabolism of ent-13(S)-hydroxyatisenoic acid and atisenoic acid by B1-41a is also described.     

Evidence for the incorporation of a fluorescent anthracene fatty acid into the membrane lipids of Micrococcus luteus

9-(2-Anthryl)-nonanoic acid, a newly synthesized photoactivable molecule, is shown to be incorporated into the membrane lipids of the bacterium Micrococcus luteus, through the regular metabolic pathway. This incorporation, which occurs at the sn-1 position exclusively and without any degradation or elongation of the anthracene fatty acid, is accompanied by an upward shift of the chain length of the other fatty acids.     

Flavour biogenesis. Partial purification and properties of a fatty acid hydroperoxide cleaving enzyme from fruits of cucumber

A membrane-bound enzyme, which catalyses the cleavage of fatty acid hydroperoxides to carbonyl fragments, has been partially purified from cucumber fruit. The isomeric 9- and 13-hydroperoxydienes (but not the hydroxydienes) derived from both linoleic and linolenic acids are cleaved by the enzyme but a mixture of 9- and 10-hydroperoxymonoenoic derivatives of oleic acid was not attacked. No evidence was obtained for free intermediates between fatty acid hydroperoxides and the cleavage products. Major volatile products were: cis-3-nonenal and hexanal (from 9- and 13-hydroperoxides of linoleic acid respectively) or cis-3,cis-6-nonadienal and cis-3-hexenal (from 9- and 13-hydroperoxides of linolenic acid). The increase in the ratio of cis-3- to trans-2-enal products with enzyme purification indicated that cis-3-enals are the immediate cleavage products and that the trans-2- forms are produced by subsequent isomerization.     

One-pot synthesis of bioactive cyclopentenones from α-linolenic acid and docosahexaenoic acid

Oxidation products of the poly-unsaturated fatty acids (PUFAs) arachidonic acid, α-linolenic acid and docosahexaenoic acid are bioactive in plants and animals as shown for the cyclopentenones prostaglandin 15d-PGJ₂ and PGA₂, cis-(+)-12-oxophytodienoic acid (12-OPDA), and 14-A-4 neuroprostane. In this study an inexpensive and simple enzymatic multi-step one-pot synthesis is presented for 12-OPDA, which is derived from α-linolenic acid, and the analogous docosahexaenoic acid (DHA)-derived cyclopentenone [(4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl]-cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid, OCPD]. The three enzymes utilized in this multi-step cascade were crude soybean lipoxygenase or a recombinant lipoxygenase, allene oxide synthase and allene oxide cyclase from Arabidopsis thaliana. The DHA-derived 12-OPDA analog OCPD is predicted to have medicinal potential and signaling properties in planta. With OCPD in hand, it is shown that this compound interacts with chloroplast cyclophilin 20-3 and can be metabolized by 12-oxophytodienoic acid reductase (OPR3) which is an enzyme relevant for substrate bioactivity modulation in planta.     

Caffeic acid derivatives isolated from the aerial parts of Galinsoga parviflora and their effect on inhibiting oxidative burst in human neutrophils

Four new caffeoyl -glucaric and -altraric acid derivatives along with eleven known compounds were isolated from aerial parts of Galinsonga parviflora. Their structures were elucidated by high-resolution spectroscopic studies. The four new compounds were determined as being 2,3,4,5-tetracaffeoylglucaric acid (1), 2,4,5-tricaffeoylglucaric acid (2), 2,3,4- or 3,4,5-tricaffeoylaltraric acid (3) and 2,3(4,5)-dicaffeoylaltraric acid (4). A reliable criterion for the determination of the linkage position of caffeic acids moieties in glucaric acid derivatives has been proposed, on the basis of detailed analysis of the respective J-couplings, including substitution and solvent influence on the observed values. All hexaric acids derivatives appeared as inhibitors of reactive oxygen species production by stimulated neutrophils.     

Generation of fad2 transgenic mice that produce omega-6 fatty acids

Fatty acid desaturase-2 (FAD2) introduces a double bond in position Δ12 in oleic acid (18︰1) to form linoleic acid (18︰2 n-6) in higher plants and microbes. A new transgenic expression cassette, containing CMV promoter/fad2 cDNA/SV40 polyA, was constructedto produce transgenic mice. Among 63 healthy offspring, 10 founders (15.9%) integrated the cotton fad2 transgene into their genomes, as demonstrated by PCR and Southern blotting analysis. All founder mice were fertile and heterozygous fad2 female and nontransgenic littermates were used for fatty acid analysis using gas chromatography. One fad2 transgenic line showed substantial differences in the fatty acid profiles and the level of linoleic acid was increased 19% (P<0.05) in transgenic muscles compared to their nontransgenic littermates. Moreover, it exhibited an 87% and a 9% increase (P<0.05) in arachidonic acid (20︰4 n-6) in muscles and liver, compared to their nontransgenic littermates. The results indicate that the plant fad2 gene can be functionally expressed in transgenic mice and may playan active role in conversion of oleic acid into linoleic acid.     

A novel abscisic acid metabolite from seeds of Robinia pseudacacia

Abscisic acid and its novel metabolise, which was a conjugated form of hydroxyabscisic acid (Metabolite C), were isolated from seeds of Robinia pseudacacia L. The structure of the conjugate was shown to be (+)-3-methyl-5 - [1(S),6(R) - 2,6 - dimethyl - 1 - hydroxy - 6 - (3 - hydroxy - 3 - methyl - 4 - carboxybutanoyloxymethyl) - 4 - oxo-cyclohex-2-enyl]-2-Z-4-E-pentadienoic acid and tentatively named β-hydroxy-β-methylglutarylhydroxyabscisic acid.