Elicitor induction of a microsomal 5-O-(4-coumaroyl)shikimate 3'-hydroxylase in parsley cell suspension cultures

Microsomal preparations from parsley cell suspension cultures challenged with an elicitor from Phytophthora megasperma f.sp. glycinea (Pmg) catalyze the formation of trans-5-O-caffeoylshikimate from trans-5-O-(4-coumaroyl)shikimate. Neither the cis isomer nor free 4-coumarate, 4-coumaroyl-CoA, or 5-O-(4-coumaroyl)quinate are substrates for this enzyme. The reaction is strictly dependent on NADPH as a reducing cofactor and on molecular oxygen. NADH, ascorbic acid, and 6,7-dimethyl-5,6,7,8-tetrahydropterine cannot substitute for NADPH. However, NADH enhances enzyme activity observed in the presence of NADPH. Cytochrome c and carbon monoxide inhibit the hydroxylation reaction, suggesting a cytochrome P-450-dependent mixed-function monooxygenase.     

Measurement of feruloyl/p-coumaroyl esterase by capillary zone electrophoresis

Ferulic andp-coumaric acid can be separated from their corresponding aliphatic methyl esters by capillary zone electrophoresis, which allows the convenient determination of feruloyl andp-coumaroyl esterase activities using synthetic esters as substrates. A feruloyl-containing sugar ester from wheat bran was also efficiently separated and used as substrate for the enzyme assays.Penicillium expansum was shown to produce feruloyl/p-coumaroyl esterase activity when grown on wheat bran in solid-state culture.The authors are with the Food Microbiology Research Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5PX, UK; A.M. McKay is also affiliated with the Department of Food Science (Microbiology), The Queen's University of Belfast, Newforge Lane, Belfast BT9 5PX, UK.     

Formation of trans-caffeoyl-CoA from trans-4-coumaroyl-CoA by Zn2+-dependent enzymes in cultured plant cells and its activation by an elicitor-induced pH shift

A novel hydroxylase activity catalyzing the formation of trans-caffeoyl-CoA from trans-4-coumaroyl-CoA was identified in crude extracts from cultured parsley cells. The extracts were less active (Vmax/Km) in converting trans-4-coumaric to trans-caffeic acid. Optimal hydroxylase activity was found at pH 6.5 with a steep decline toward both pH 7.4 and pH 5.0. The enzyme activity requires ascorbate and Zn2+ at optimal concentrations of 50 and 0.5 mM, respectively. No other reductant could replace ascorbate, whereas high concentrations of Ca2+ partially substituted for Zn2+. The enzyme is soluble and appears to be located in the cytoplasm. The unusual pH optimum suggests that the hydroxylase is inactive at the normal cytoplasmic pH. Upon treatment of parsley cells with an elicitor derived from Phytophthora megasperma f. sp. glycinea, the cytoplasmic pH dropped by approximately 0.25 pH unit within 55 min as determined by 31P NMR spectroscopy. Our results suggest that this shift in the cytoplasmic pH is sufficient for the activation of the hydroxylase, eventually leading to the formation of caffeoyl and feruloyl esters. Such esters may be a part of a very rapid resistance response of the plant cells, which would leave no time for de novo enzyme synthesis.     

Monolignol biosynthesis in microsomal preparations from lignifying stems of alfalfa (Medicago sativa L.)

Microsomal preparations from lignifying stems of alfalfa (Medicago sativa L.) contained coniferaldehyde 5-hydroxylase activity and immunodetectable caffeic acid 3-O-methyltransferase (COMT), and catalyzed the S-adenosyl L-methionine (SAM) dependent methylation of caffeic acid, caffeyl aldehyde and caffeyl alcohol. When supplied with NADPH and SAM, the microsomes converted caffeyl aldehyde to coniferaldehyde, 5-hydroxyconiferaldehyde, and traces of sinapaldehyde. Coniferaldehyde was a better precursor of sinapaldehyde than was 5-hydroxyconiferaldehyde. The alfalfa microsomes could not metabolize 4-coumaric acid, 4-coumaraldehyde, 4-coumaroyl CoA, or ferulic acid. No metabolism of monolignol precursors was observed in microsomal preparations from transgenic alfalfa down-regulated in COMT expression. In most microsomal preparations, the level of the metabolic conversions was independent of added recombinant COMT. Taken together, the data provide only limited support for the concept of metabolic channeling in the biosynthesis of S monolignols via coniferaldehyde.     

Purification,cloning, and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism

A protein hydrolyzing hydroxycinnamoyl-CoA esters has been purified from tobacco stem extracts by a series of high pressure liquid chromatography steps. The determination of its N-terminal amino acid sequence allowed design of primers permitting the corresponding cDNA to be cloned by PCR. Sequence analysis revealed that the tobacco gene belongs to a plant acyltransferase gene family, the members of which have various functions. The tobacco cDNA was expressed in bacterial cells as a recombinant protein fused to glutathione S-transferase. The fusion protein was affinity-purified and cleaved to yield the recombinant enzyme for use in the study of catalytic properties. The enzyme catalyzed the synthesis of shikimate and quinate esters shown recently to be substrates of the cytochrome P450 3-hydroxylase involved in phenylpropanoid biosynthesis. The enzyme has been named hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyltransferase. We show that p-coumaroyl-CoA and caffeoyl-CoA are the best acyl group donors and that the acyl group is transferred more efficiently to shikimate than to quinate. The enzyme also catalyzed the reverse reaction, i.e. the formation of caffeoyl-CoA from chlorogenate (5-O-caffeoyl quinate ester). Thus, hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase appears to control the biosynthesis and turnover of major plant phenolic compounds such as lignin and chlorogenic acid.     

3, 4-Dihydroxyphenylalanine is oxidized by phenoxyl radicals of hydroxycinnamic acid esters in leaves ofVicia faba L

Mechanisms of oxidation of 3,4-dihydroxyphenylalanine (dopa) in leaves ofVicia faba have not yet been elucidated in details. The author hypothesized its oxidation by radicals of hydroxycinnamic acid esters that were generated by a peroxidase-dependent reaction in vacuoles. The results obtained in this study were followings. 1) Vacuolar peroxidase isolated from the leaves oxidized dopa more slowly than 4-coumaric and caffeic acid esters isolated from the leaves. 2) The hydroxycinnamic acid esters enhanced peroxidase-dependent oxidation of dopa and dopa suppressed their oxidation. 3) Degree of the enhancement was roughly correlated with rates of the oxidation of hydroxycinnamic acid esters. 4) The hydroxycinnamic acid esters increased levels of dopa radical in the presence of peroxidase. 5) In protoplasts of mesophyll cells ofV. faba, hydrogen peroxide-induced oxidation of dopa was faster than that of 4-coumaric acid and caffeic acid esters. These results support the above hypothesis that dopa in vacuoles is oxidized by phenoxyl radicals of hydroxycinnamic acid esters that are generated by vacuolar peroxidase.     

Purification and characterization of a feruloyl esterase from the fungus Penicilliumexpansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and ρ-coumaric acid from methyl esters of theacids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose (FAXX) and O-[5-O-((E)-ρ-coumaroyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose(PAXX). The esterase was purified 360-fold in successive stepsinvolving ultrafiltration and column chromatography by gel filtration, anion exchange andhydrophobic interaction. These chromatographic methods separated the phenolic acid esterasefrom α- l -arabinofuranosidase, pectate and pectin lyase, polygalacturonase,xylanase and β- d -xylosidase activities. The phenolic acid esterase had an apparentmass of 65 kDa under non-denaturing conditions and a mass of 57·5 kDa underdenaturing conditions. Optimal pH and temperature were 5·6 and 37 °C,respectively and the metal ions Cu2+ and Fe³+ atconcentrations of 5 mmol l⁻¹ inhibited feruloyl esterase activity by 95% and44%, respectively, at the optimum pH and temperature. The apparent K_m and V_max of the purified feruloyl esterase for methyl ferulate at pH 5·6 and 37 °Cwere 2·6 mmol l⁻¹ and 27·1 μmol min⁻¹ mg⁻¹. The corresponding constants of ρ-coumaroylesterase for methyl coumarate were 2·9 mmol l⁻¹ and 18·6μmol min−1 mg⁻¹.     

Acylated pelargonidin glycosides in the red-purple flowers of Pharbitis nil.

Four acylated pelargonidin glycosides and pelargonidin 3-sophoroside-5-glucoside were isolated from 23 red-purple cultivars of Pharbitis nil. The acylated anthocyanins were all based on pelargonidin 3-sophoroside-5-glucoside and were identified as the 3-O-[2-O-(beta-D-glucopyranosyl)-6-O-(trans-caffeyl)-beta-D- glucopyranoside]-5-O-(beta-D-glucopyranoside), the 3-O-[2-O-(6-O-(trans-3-O-(beta-D-glucopyranosyl)caffeyl)-beta- D-glucopyranosyl)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside), the 3-O-[2-O-(6-O-(trans-3-O-(beta-D-glucopyranosyl)caffeyl)-beta- D-glucopyranosyl)-6-O-(trans-caffeyl)-beta-D-glucopyranoside]-5-O-(beta- D-glucopyranoside); and the 3-O-[2-O-(6-O-(trans-3-O-(beta-D-glucopyranosyl)caffeyl)-beta-D- glucopyranosyl)-6-O-(trans-4-O-(6-O-(trans-3-O-(beta-D- glucopyranosyl)caffeyl)- beta-D-glucopyranosyl)caffeyl)-beta-D-glucopyranoside]-5-O-(beta-D- glucopyranoside). By the analysis of these anthocyanin constituents variously in 23 cultivars, it was found that the red flower colour gradually changed into more bluish colour with increasing numbers of caffeic acid residues in the acylated pelargonidin glycosides. The stabilities of these anthocyanins increased in the order of increasing caffeyl substitution.     

Mitochondrial trans-2-enoyl-CoA reductase of wax ester fermentation from Euglena gracilis defines a new family of enzymes involved in lipid synthesis

Under anaerobiosis, Euglena gracilis mitochondria perform a malonyl-CoA independent synthesis of fatty acids leading to accumulation of wax esters, which serve as the sink for electrons stemming from glycolytic ATP synthesis and pyruvate oxidation. An important enzyme of this unusual pathway is trans-2-enoyl-CoA reductase (EC 1.3.1.44), which catalyzes reduction of enoyl-CoA to acyl-CoA. Trans-2-enoyl-CoA reductase from Euglena was purified 1700-fold to electrophoretic homogeneity and was active with NADH and NADPH as the electron donor. The active enzyme is a monomer with molecular mass of 44 kDa. The amino acid sequence of tryptic peptides determined by electrospray ionization mass spectrometry were used to clone the corresponding cDNA, which encoded a polypeptide that, when expressed in Escherichia coli and purified by affinity chromatography, possessed trans-2-enoyl-CoA reductase activity close to that of the enzyme purified from Euglena. Trans-2-enoyl-CoA reductase activity is present in mitochondria and the mRNA is expressed under aerobic and anaerobic conditions. Using NADH, the recombinant enzyme accepted crotonyl-CoA (km=68 microm) and trans-2-hexenoyl-CoA (km=91 microm). In the crotonyl-CoA-dependent reaction, both NADH (km=109 microm) or NADPH (km=119 microm) were accepted, with 2-3-fold higher specific activities for NADH relative to NADPH. Trans-2-enoyl-CoA reductase homologues were not found among other eukaryotes, but are present as hypothetical reading frames of unknown function in sequenced genomes of many proteobacteria and a few Gram-positive eubacteria, where they occasionally occur next to genes involved in fatty acid and polyketide biosynthesis. Trans-2-enoyl-CoA reductase assigns a biochemical activity, NAD(P)H-dependent acyl-CoA synthesis from enoyl-CoA, to one member of this gene family of previously unknown function.     

Differential effects of light and fungal elicitor on chlorogenic acid and caffeoylshikimic acid metabolism

Upon irradiation an increase in the extractable activity of hydroxycinnamoyl-CoA:d-quinate hydroxycinnamoyl transferase (CQT) and a decrease in the activity of hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (CST) was observed in cell suspension cultures and seedling of carrot (Daucus carota L). Conversely, CST was induced and CQT repressed in the cell cultures upon treatment with fungal elicitor (i.e. cell wall preparations from Phytophthora megasperma). In the cell cultures irradiation led to a continuous accumulation of 5-O-caffeoyl-d-quinic (chlorogenic) acid, while a transient accumulation of 5-O-caffeoylshikimic acid took place in response to elicitor treatment. Cell wall bound 4-hydroxybenzoic, 4-coumaric and also ferulic acid were increased after treatment with Pmg elicitor. These wall bound phenolics may be involved in protection against microbil attack.     

The mechanism of dienoyl-CoA reduction by 2,4-dienoyl-CoA reductase is stepwise: observation of a dienolate intermediate

The chemical mechanism of the 2,4-dienoyl-CoA reductase (EC 1.3.1.34) from rat liver mitochondria has been investigated. This enzyme catalyzes the NADPH-dependent reduction of 2,4-dienoyl-coenzyme A (CoA) thiolesters to the resulting trans-3-enoyl-CoA. Steady-state kinetic parameters for trans-2,trans-4-hexadienoyl-CoA and 5-phenyl-trans-2,trans-4-pentadienoyl-CoA were determined and demonstrated that the dienoyl-CoA and NADPH bind to the 2,4-dienoyl-CoA reductase via a sequential kinetic mechanism. Kinetic isotope effect studies and the transient kinetics of substrate binding support a random order of nucleotide and dienoyl-CoA addition. The large normal solvent isotope effects on V/K ((D)(2)(O)V/K) and V ((D)(2)(O)V) for trans-2,trans-4-hexadienoyl-CoA reduction indicate that a proton transfer step is rate limiting for this substrate. The stability gained by conjugating the phenyl ring to the diene in PPD-CoA results in the reversal of the rate-determining step, as evidenced by the normal isotope effects on V/K(CoA) ((D)V/K(CoA)) and V/K(NADPH) ((D)V/K(NADPH)). The reversal of the rate-determining step was supported by transient kinetics where a burst was observed for the reduction of trans-2,trans-4-hexadienoyl-CoA but not for 5-phenyl-trans-2,trans-4-pentadienoyl-CoA reduction. The chemical mechanism is stepwise where hydride transfer from NADPH occurs followed by protonation of the observable dienolate intermediate, which has an absorbance maximum at 286 nm. The exchange of the C alpha protons of trans-3-decenoyl-CoA, catalyzed by the 2,4-dienoyl-CoA reductase, in the presence of NADP(+) suggests that formation of the dienolate is catalyzed by the enzyme active site.     

Crystal structure of a novel shikimate dehydrogenase from Haemophilus influenzae

To date two classes of shikimate dehydrogenases have been identified and characterized, YdiB and AroE. YdiB is a bifunctional enzyme that catalyzes the reversible reductions of dehydroquinate to quinate and dehydroshikimate to shikimate in the presence of either NADH or NADPH. In contrast, AroE catalyzes the reversible reduction of dehydroshikimate to shikimate in the presence of NADPH. Here we report the crystal structure and biochemical characterization of HI0607, a novel class of shikimate dehydrogenase annotated as shikimate dehydrogenase-like. The kinetic properties of HI0607 are remarkably different from those of AroE and YdiB. In comparison with YdiB, HI0607 catalyzes the oxidation of shikimate but not quinate. The turnover rate for the oxidation of shikimate is approximately 1000-fold lower compared with that of AroE. Phylogenetic analysis reveals three independent clusters representing three classes of shikimate dehydrogenases, namely AroE, YdiB, and this newly characterized shikimate dehydrogenase-like protein. In addition, mutagenesis studies of two invariant residues, Asp-103 and Lys-67, indicate that they are important catalytic groups that may function as a catalytic pair in the shikimate dehydrogenase reaction. This is the first study that describes the crystal structure as well as mutagenesis and mechanistic analysis of this new class of shikimate dehydrogenase.     

Formation of hydrogen peroxide by isolated cell walls from horseradish (Armoracia lapathifolia Gilib.)

Summary Isolated cell-wall suspensions from horseradish in the presence of 5×10^-4 M MnCl₂ catalyze the production of hydrogen peroxide at the expense of either NADPH or NADH. This reaction is inhibited by scavengers of the superoxide free radical ion such as ascorbate or dihydroxyphenols or by superoxide dismutase, and stimulated by monophenols such as p-coumaric acid. On comparison with isolated (commercial) horseradish peroxidase it becomes evident that (a) cell-wall-bound peroxidase(s) is (are) responsible for the production of hydrogenperoxide, involving the superoxide free radical ion as an intermediate of the complex reaction chain.Abbreviation SOD superoxide dismutase     

Expression of bacterial tyrosine ammonia-lyase creates a novel p-coumaric acid pathway in the biosynthesis of phenylpropanoids in Arabidopsis

Some flavonoids are considered as beneficial compounds because they exhibit anticancer or antioxidant activity. In higher plants, flavonoids are secondary metabolites that are derived from phenylpropanoid biosynthetic pathway. A large number of phenylpropanoids are generated from p-coumaric acid, which is a derivative of the primary metabolite, phenylalanine. The first two steps in the phenylpropanoid biosynthetic pathway are catalyzed by phenylalanine ammonia-lyase and cinnamate 4-hydroxylase, and the coupling of these two enzymes forms a rate-limiting step in the pathway. For the generation of p-coumaric acid, the conversion from phenylalanine to p-coumaric acid that is catalyzed by two enzymes can be theoretically performed by a single enzyme, tyrosine ammonia-lyase (TAL) that catalyzes the conversion of tyrosine to p-coumaric acid in certain bacteria. To modify the p-coumaric acid pathway in plants, we isolated a gene encoding TAL from a photosynthetic bacterium, Rhodobacter sphaeroides, and introduced the gene (RsTAL) in Arabidopsis thaliana. Analysis of metabolites revealed that the ectopic over-expression of RsTAL leads to higher accumulation of anthocyanins in transgenic 5-day-old seedlings. On the other hand, 21-day-old seedlings of plants expressing RsTAL showed accumulation of higher amount of quercetin glycosides, sinapoyl and p-coumaroyl derivatives than control. These results indicate that ectopic expression of the RsTAL gene in Arabidopsis enhanced the metabolic flux into the phenylpropanoid pathway and resulted in increased accumulation of flavonoids and phenylpropanoids.     

Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3-oxo, 4-oxo and 5-oxo esters in baker's yeast

Two NADPH-dependent oxidoreductases catalyzing the enantioselective reduction of 3-oxo esters to (S)- and (R)-3-hydroxy acid esters, [hereafter called (S)- and (R)-enzymes] have been purified 121- and 332-fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, Sephadex G-150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography. The relative molecular mass Mr, of the (S)-enzyme was estimated to be 48,000-50,000 on Sephadex G-150 column chromatography and 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and reduced 3-oxo esters, 4-oxo and 5-oxo acids and esters enantioselectively to (S)- hydroxy compounds in the presence of NADPH. The Km values for ethyl 3-oxobutyrate, ethyl 3-oxohexanoate, 4-oxopentanoic and 5-oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively. The Mr of the (R)-enzyme, estimated by means of column chromatography on Sepharose 6B, was 800,000. Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200,000 and 210,000, respectively. The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3-oxo esters to (R)-hydroxy esters, using NADPH for coenzyme. Km values for ethyl 3-oxobutyrate and ethyl 3-oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively. Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)-enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr 2.4 X 10(6)) showed no activity in catalyzing the reduction of 3-oxo esters.     

Isolation and characterization of sialate 9(4)-O-acetylesterase from influenza C virus

An esterase was isolated from influenza C virus with a specific activity from 1.7-5 U/mg protein, and its substrate specificity was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. The enzyme hydrolyses only acetic acid esters at significant rates. The non-natural substrates 4-methyl-umbelliferyl acetate, 4-nitrophenyl acetate, and alpha-naphthyl acetate are cleaved at highest hydrolysis rates, followed by the natural substrate N-acetyl-9-O-acetylneuraminic acid. The esterase also acts on N-glycoloyl-9-O-acetylneuraminic acid and, much slower, on N-acetyl-4-O-acetylneuraminic acid; N-acetyl-7-O-acetylneuraminic acid is not hydrolysed. 2-Deoxy-2,3-didehydro-N-acetyl-9-O-acetylneuraminic acid is also a substrate for this enzyme, however, 6-O-acetylated N-acetylmannosamine and glucose are not. Esterification of the carboxyl function of sialic acids strongly reduces or prevents esterase action on O-acetyl groups. The carboxyl ester is not hydrolysed. The relative cleavage rates also depend on the type of the non-sialic acid part of the molecule. N-Acetyl-9-O-acetylneuraminic acid as component of sialyllactose and rat serum glycoprotein shows hydrolysis rates close to the free form of this sugar, while acetyl ester groups of bovine submandibular gland mucin and rat erythrocytes are hydrolysed at slower rates. Gangliosides and 4-O-acetylated glycoproteins are no substrates for the purified enzyme. A slow hydrolysis is observed by incubation of 9-O-acetylated GD1a with intact influenza C viruses. As other natural acetyl esters (acetyl-CoA and acetylthiocholine iodide) are not hydrolysed, the enzyme can be classified as sialate 9(4)-O-acetylesterase (EC 3.1.1.53).     

Vacuolar localization of the enzymatic synthesis of hydroxycinnamic acid esters of malic acid in protoplasts from Raphanus sativus leaves

Protoplasts from leaves of radish ( Raphanus sativus L. var. sativus ) were examined for the subcellular localization of p -coumaric, caffeic, ferulic and sinapic acid esters of malic acid and the enzyme(s) involved in their syntheses. Vacuoles isolated from leaf protoplasts contained all the hydroxycinnamic acid esters as well as all the dependent enzyme activities. Protein from leaf vacuoles was shown to form the hydroxycinnamoylmalic acids, using the corresponding hydroxycinnamic acid glucose esters (1-O-acyl glucosides) as acyl donors. It is proposed that the vacuole is the cell compartment for synthesis and deposition of the hydroxycinnamoylmalic acids.     

An Aspergillus niger esterase (ferulic acid esterase III) and a recombinant Pseudomonas fluorescens subsp. cellulosa esterase (Xy1D) release a 5-5' ferulic dehydrodimer (diferulic acid) from barley and wheat cell walls.

Diferulate esters strengthen and cross-link primary plant cell walls and help to defend the plant from invading microbes. Phenolics also limit the degradation of plant cell walls by saprophytic microbes and by anaerobic microorganisms in the rumen. We show that incubation of wheat and barley cell walls with ferulic acid esterase from Aspergillus niger (FAE-III) or Pseudomonas fluorescens (Xy1D), together with either xylanase I from Aspergillus niger, Trichoderma viride xylanase, or xylanase from Pseudomonas fluorescens (XylA), leads to release of the ferulate dimer 5-5' diFA [(E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid]. Direct saponification of the cell walls without enzyme treatment released the following five identifiable ferulate dimers (in order of abundance): (Z)-beta-(4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy)-4-hydroxy-3-methoxycinnamic acid, trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxy-phenyl) -7-methoxy-2, 3-dihydrobenzofuran-3-carboxylic acid, 5-5' diFA, (E,E)-4, 4'-dihydroxy-3, 5'-dimethoxy-beta, 3'-bicinnamic acid, and trans-7-hydroxy-1-(4-hydroxy-3-methoxyphenyl) -6-methoxy-1, 2-dihydronaphthalene-2, 3-dicarboxylic acid. Incubation of the wheat or barley cell walls with xylanase, followed by saponification of the solubilized fraction, yielded 5-5'diFA and, in some cases, certain of the above dimers, depending on the xylanase used. These experiments demonstrate that FAE-III and XYLD specifically release only esters of 5-5'diFA from either xylanase-treated or insoluble fractions of cell walls, even though other esterified dimers were solubilized by preincubation with xylanase. It is also concluded that the esterified dimer content of the xylanase-solubilized fraction depends on the source of the xylanase.     

Synthesis and glucose-6-phosphatase inhibitory activity of (thiouriedo)alkanoic acid esters

A series of (3-pyridin-2-yl-thiouriedo)alkanoic acid esters (5a-j) have been synthesized by the reaction of pyridin-2-yl-dithiocarbamic acid methyl ester (2) and amino acid esters (4). Most of the synthesized compounds have been evaluated against glucose-6-phosphatase enzyme but only four compounds (5g-j) displayed significant inhibitory activity of the enzyme.