Metabolism of aromatic acids by Polyporus hispidus

When grown on glucose as principal carbon source the culture medium of Polyporus hispidus was found to contain phenolic acids, including p-coumaric and caffeic acids. ¹⁴C-Studies indicated that phenylalanine is converted to cinnamic acid as well as to phenylpyruvic acid and tyrosine in cultures. Cell-free preparations of mycelium contained phenylalanine and tyrosine ammonia-lyse activities and were capable of effecting the hydroxylation of cinnamic, p-coumaric and benzoic acids.     

Metabolism of Aromatic Amino Acids in Microorganisms

It was found that when Rhodotorula rubra IFO 0911 was grown in a phenylalanine medium, benzoic acid and p-hydroxybenzoic acid besides cinnamic acid were formed in the cultured both. The conversions of cinnamic acid into benzoic acid and of benzoic acid into p-hydroxybenzoic acid, and the degradation of p-hydroxybenzoic acid were demonstrated in intact cells of Rhodotorula rubra. These activities were observed in the cells grown on various media, including the medium containing no phenylalanine, and were found to be distributed widely in Rhodotorula. The cells of Rhodotorula rubra were also able to degrade p-coumaric acid, 3,4-dihydroxybenzoic acid (protocatechuic acid), p-hydroxyphenyl-acetic acid, 3-methoxy-4-hydroxycinnamic acid (ferulic acid) and 3-methoxy-4-hydroxybenzoic acid (vanillic acid). From these results, the metabolic pathways for phenylalanine and tyrosine in Rhodotorula were discussed.     

Soil phenolics and plant growth inhibition

Summary Vanillic acid, p-hydroxy benzoic acid, p-coumaric acid and three other unidentified phenolic acids were detected in the Annamalainagar rice field soils. The quantity of total phenols decreased significantly following increased dose of nitrogenous fertilizer. The rice cultivar Co. 13 responded well to increasing N application. When tested in vitro, cinnamic acid even at 0.0001 M concentration proved detrimental to the growth of rice seedlings. The decrease in the level of phenols in soil following increased N application was suggested as one of the causes for prolific growth of rice plants. re]19721024     

Regulation of phenylpropanoid metabolism by exogenous precursors in axenic cultures of Sphagnum fallax

Sphagnum plantlets, cultivated in continuous-feed bioreactors, are characterised by high levels of free endogenous phenolics and a pronounced excretion of some phenolics into the effluent culture medium. The transfer of Sphagnum fallax, precultivated in continuous-feed bioreactors, to batch cultures resulted in an increased flux through phenylpropanoid metabolism and an accumulation of p-coumaric acid to 0.1 μM and of trans-sphagnum acid up to 0.5 μM in the external medium [³H]-labelled L-phenylalanine (7.7 GBq mol^?1) was rapidly taken up, resulting in an enhanced synthesis and excretion of p-coumaric and trans-sphagnum acid. Specific activities were 6.9 and 5.4 GBq mol^?1, respectively, for these cinnamic acids excreted into the external medium. Endogenous pools of trans-cinnamic and p-coumaric acid did not increase and no labelling could be detected in these compounds. Cell wall-bound activity amounted to ca 14% of the applied activity after 48 h of incubation, 59% of which was recovered in dioxane/2 M HCl extracts of the cell wall. Exogenously applied trans-cinnamic acid (0.1 mM) was taken up to 46% and resulted in a transient endogenous accumulation of trans-cinnamic acid, the level of free endogenous p-coumaric and trans-sphagnum acid was found to have decreased. The concentrations of p-coumaric and trans-sphagnum acid in the culture medium rose to 17 and 2.4 μM, respectively, after 48 h of incubation in 0.1 mMtrans-cinnamic acid. Exogenously applied p-coumaric acid (0.1 mM) was taken up to 79% from the incubation solution but not stored endogenously, as metabolic products trans-sphagnum acid and an unknown p-coumaric acid-conjugate accumulated in the external medium and endogenously. These results give evidence for the biosynthetical route from phenylalanine to sphagnum acid and a channelling of pathway intermediates by the enzymes L-phenylalanine ammonia-lyase (EC 4.3.1.5) and cinnamic acid 4-hydroxylase (EC 1.14.13.11).     

Isolation and Characterization of Thermophilic Bacilli Degrading Cinnamic, 4-Coumaric, and Ferulic Acids

Thirty-four thermophilic Bacillus sp. strains were isolated from decayed wood bark and a hot spring water sample based on their ability to degrade vanillic acid under thermophilic conditions. It was found that these bacteria were able to degrade a wide range of aromatic acids such as cinnamic, 4-coumaric, 3-phenylpropionic, 3-(p-hydroxyphenyl)propionic, ferulic, benzoic, and 4-hydroxybenzoic acids. The metabolic pathways for the degradation of these aromatic acids at 60°C were examined by using one of the isolates, strain B1. Benzoic and 4-hydroxybenzoic acids were detected as breakdown products from cinnamic and 4-coumaric acids, respectively. The β-oxidative mechanism was proposed to be responsible for these conversions. The degradation of benzoic and 4-hydroxybenzoic acids was determined to proceed through catechol and gentisic acid, respectively, for their ring fission. It is likely that a non-β-oxidative mechanism is the case in the ferulic acid catabolism, which involved 4-hydroxy-3-methoxyphenyl-β-hydroxypropionic acid, vanillin, and vanillic acid as the intermediates. Other strains examined, which are V0, D1, E1, G2, ZI3, and H4, were found to have the same pathways as those of strain B1, except that strains V0, D1, and H4 had the ability to transform 3-hydroxybenzoic acid to gentisic acid, which strain B1 could not do.     

Degradation of phenylalanine and tyrosine by Sporobolomyces roseus

Ammonia-lyase activity for l-phenylalanine, m-hydroxyphenylalanine and l-tyrosine was demonstrated in cell-free extracts of Sporobolomyces roseus. Cultures of this organism converted dl-[ring-¹⁴C]phenylalanine and l-[U-¹⁴C]tyrosine into the corresponding cinnamic acid. Tracer studies showed that these compounds were further metabolized to [¹⁴C]protocatechuic acid. Benzoic acid and p-hydroxybenzoic acid were intermediates in this pathway. Washed cells of the organism readily utilized cinnamic acid, p-coumaric acid, caffeic acid, benzoic acid and p-hydroxybenzoic acid. Protocatechuic acid was the terminal aromatic compound formed during the metabolism of these compounds. The cells of S. roseus were able to convert m-coumaric acid into m-hydroxybenzoic acid, but the latter compound, which accumulated in the medium, was not further metabolized. 4-Hydroxycoumarin was identified as the product of o-coumaric acid metabolism by this organism.     

The enzymic conversion of hydroxycinnamic acids to p-coumarylquinic and chlorogenic acids in tomato fruits

Two enzymes thought to be involved in the biosynthesis of chlorogenic acid have been separated and purified by ion exchange chromatography and their properties studied. These two enzymes, p-coumarate CoA ligase and hydroxycinnamyl CoA: quinate hydroxycinnamyl transferase, acting together catalyse the conversion of p-coumaric acid to 5′-p-coumarylquinic acid and of caffeic acid to chlorogenic acid. The ligase has a higher affinity for p-coumaric than for caffeic acid and will in addition activate a number of other cinnamic acids such as ferulic, isoferulic and m-coumaric acids but not cinnamic acid. The transferase shows higher activity and affinity with p-coumaryl CoA than caffeyl CoA. It also acts with ferulyl CoA but only very slowly. The enzyme shows high specificity for quinic acid; shikimic acid is esterified at only 2% of the rate with quinic acid and glucose is not a substrate. The transferase activity is reversible and both chlorogenic acid and 5′-p-coumarylquinic acids are cleaved in the presence of CoA to form quinic acid and the corresponding hydroxycinnamyl CoA thioester.     

Further studies on the biosynthesis of mangiferin in Anemarrhena asphodeloides: hydroxylation of the shikimate-derived ring

The hydroxylation at C-3′ of maclurin, an intermediate in mangiferin biosynthesis, has been studied. Labelled cinnamic acid, p-coumaric acid, caffeic acid, iriflophenone and maclurin were fed to Anemarrhena asphodeloides. Cinnamic acid and p-coumaric acid were better precursors than caffeic acid for mangiferin, and iriflophenone as well as maclurin was effectively incorporated into mangiferin and isomangiferin. These results show that maclurin is biosynthesized via hydroxylation of iriflophenone derived from p-coumarate in this plant.     

Enzymatic Determination of l-Phenylalanine and l-Tyrosine

An enzymatic method for the determination of phenylalanine and tyrosine has been described. This method is based on the formation of cinnamic acid from phenylalanine or the formation of p-coumaric acid from tyrosine by phenylalanine ammonia-lyase of Rhodotorula. Cinnamic acid and p-coumaric acid, which are formed in stoichiometric amounts, are determined spectrophotometrically. Other amino acids and d-isomers of phenylalanine and tyrosine have no effect on this determination.     

Possible pathways for destruction of polyaromatic hydrocarbons by some oil-degrading bacteria isolated from plant endosphere and rhizosphere

Six strains of oil-degrading bacteria isolated from the endosphere and rhizosphere of plants growing on oil polluted soils of the Irkutsk region were studied to determine the pathways for biodestruction of polyaromatic oil hydrocarbons. All strains were able to efficiently degrade polyaromatic hydrocarbons with the formation of pyrocatechin as a final product; strains 90, 108, and 112 additionally formed protocathechuic acid. The culture broth of the studied strains contained ferulic, n-coumaric, n-oxybenzoic, vanillic, and lilac acids, which probably represent metabolites of cinnamic alcohol, cinnamic aldehyde, and benzoic acid presenting in oil and metabolized by bacteria.     

New insights into the antioxidant activity of hydroxycinnamic and hydroxybenzoic systems: Spectroscopic,electrochemistry, and cellular studies

Abstract

A series hydroxycinnamic and gallic acids and their derivatives were studied with the aim of evaluating their in vitro antioxidant properties both in homogeneous and in cellular systems. It was concluded from the oxygen radical absorbance capacity-fluorescein (ORAC-FL), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and cyclic voltammetry data that some compounds exhibit remarkable antioxidant properties. In general, in homogeneous media (DPPH assay), galloyl-based cinnamic and benzoic systems (compounds 7–11) were the most active, exhibiting the lowest oxidation potentials in both dimethyl sulfoxide (DMSO) and phosphate buffer. Yet, p-coumaric acid and its derivatives (compounds 1–3) disclosed the highest scavenging activity toward peroxyl radicals (ORAC-FL assay). Interesting structure–property– activity relationships between ORAC-FL, or DPPH radical, and redox potentials have been attained, showing that the latter parameter can be a valuable antioxidant measure. It was evidenced that redox potentials are related to the structural features of cinnamic and benzoic systems and that their activities are also dependent on the radical generated in the assay. Electron spin resonance data of the phenoxyl radicals generated both in DMSO and phosphate buffer support the assumption that radical stability is related to the type of phenolic system. Galloyl-based cinnamic and benzoic ester-type systems (compounds 9 and 11) were the most active and effective compounds in cell-based assays (51.13 ± 1.27% and 54.90 ± 3.65%, respectively). In cellular systems, hydroxycinnamic and hydroxybenzoic systems operate based on their intrinsic antioxidant outline and lipophilic properties, so the balance between these two properties is considered of the utmost importance to ensure their performance in the prevention or minimization of the effects due to free radical overproduction.     

3-Hydroxybenzoate:coenzyme A ligase and 4-coumarate: coenzyme A ligase from cultured cells of Centaurium erythraea

3-Hydroxybenzoate:coenzyme A ligase, an enzyme involved in xanthone biosynthesis, was detected in cell-free extracts from cultured cells of Centaurium erythraea Rafn. The enzyme was separated from 4-coumarate:coenzyme A ligase by fractionated ammonium sulphate precipitation and hydrophobic interaction chromatography. The CoA ligases exhibited different substrate specificities. 3-Hydroxybenzoate:coenzyme A ligase activated 3-hydroxybenzoic acid most efficiently and lacked affinity for cinnamic acids. In contrast, 4-coumarate:CoA ligase mainly catalyzed the activation of 4-coumaric acid but did not act on benzoic acids. The two enzymes were similar with respect to their relative molecular weight, their pH and temperature optima, their specific activity and the changes in their activity during cell culture growth. Received: 23 September 1996 / Accepted: 28 November 1996     

The effect of blue light on free and esterified phenolic acids in etiolated gherkin tissues

The major phenolic acid found in gherkin tissues is p-coumaric acid, although cinnamic and caffeic acids are also present; these occur both free an     

Enhanced Lignin Monomer Production Caused by Cinnamic Acid and Its Hydroxylated Derivatives Inhibits Soybean Root Growth

Cinnamic acid and its hydroxylated derivatives (p-coumaric, caffeic, ferulic and sinapic acids) are known allelochemicals that affect the seed germination and root growth of many plant species. Recent studies have indicated that the reduction of root growth by these allelochemicals is associated with premature cell wall lignification. We hypothesized that an influx of these compounds into the phenylpropanoid pathway increases the lignin monomer content and reduces the root growth. To confirm this hypothesis, we evaluated the effects of cinnamic, p-coumaric, caffeic, ferulic and sinapic acids on soybean root growth, lignin and the composition of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) monomers. To this end, three-day-old seedlings were cultivated in nutrient solution with or without allelochemical (or selective enzymatic inhibitors of the phenylpropanoid pathway) in a growth chamber for 24 h. In general, the results showed that 1) cinnamic, p-coumaric, caffeic and ferulic acids reduced root growth and increased lignin content; 2) cinnamic and p-coumaric acids increased p-hydroxyphenyl (H) monomer content, whereas p-coumaric, caffeic and ferulic acids increased guaiacyl (G) content, and sinapic acid increased sinapyl (S) content; 3) when applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), cinnamic acid reduced H, G and S contents; and 4) when applied in conjunction with 3,4-(methylenedioxy)cinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL), p-coumaric acid reduced H, G and S contents, whereas caffeic, ferulic and sinapic acids reduced G and S contents. These results confirm our hypothesis that exogenously applied allelochemicals are channeled into the phenylpropanoid pathway causing excessive production of lignin and its main monomers. By consequence, an enhanced stiffening of the cell wall restricts soybean root growth.     

Immunocytochemical localization of phenolic compounds in pollen walls using antibodies againstp-coumaric acid coupled to bovine serum albumin

Summary Two different antibodies against bovine serum albumin (BSA)-p-coumaric acid-conjugates were produced and used to localize phenolic compounds in exines of pollen from different species,p-Coumaric acid (pC) was coupled to BSA either via the carboxy group (BSA-pC) or directly to the aromatic ring system (BSA-azopC). The polyclonal antibodies raised in rabbits were characterized by ELISA with homologous and heterologous antigens using turkey ovalbumin as carrier protein. The results showed that the two immune sera directed against BSA-pC and BSA-azo-pC, respectively, were specific forp-coumaric acid and structurally similar compounds. Only a very poor binding by acetic acid-ovalbumin-conjugates and no binding by turkey ovalbumin was detectable. The antibodies reacted with partially purified pollen walls and with highly purified exines. The intensity of the immune reaction was proved to be dependent upon the pollen source and the preparation of the pollen walls. Using light and electron microscopy, it was shown for the first time that, in the exines ofCucurbita maxima, antibody binding was predominantly observed in the region of the germ pore apertures, the outer foot layers, and in the micro- and macrospines. We conclude from this and other earlier published data that phenols are important structural compounds of sporopollenin.Abbrevations AA acetic acid - BA benzoic acid - BSA bovine serum albumin - BSA-azo-pC p-coumaric acid coupled in meta position to BSA by a diazo reaction - BSA-azo-pC I immune serum against BSA-azo-pC - BSA-pC p-coumaric acid coupled to BSA via the COOH-group - BSA-pC I immune serum against BSA-pC - FA ferulic acid - OVA ovalbunin from turkey - pC p-coumaric acid - pHY p-hydroxybenzoic acid - SA sinapic acid - SYA syringic acid - TAT TBS-azide-Tween-buffer - TFA trifluoroacetic acid - VA vanillic acid     

Co-production of caffeic acid and p-hydroxybenzoic acid from p-coumaric acid by Streptomyces caeruleus MTCC 6638

In a culture medium of Streptomyces caeruleus MTCC 6638 grown with p-coumaric acid (5 mM) as the sole source of carbon, co-production of caffeic acid and p-hydroxybenzoic acid was observed. Both caffeic acid and p-hydroxybenzoic acid are important phenolic compounds with pharmaceutical importance. These biotransformed products were identified by high-performance liquid chromatography and electrospray ionization mass spectrometry. Obtained data suggest that p-coumaric acid was possibly utilized by two different routes, resulting in the formation of a hydroxycinnamate and a hydroxybenzoate compound. However, higher concentration of p-coumaric acid (10 mM) favoured caffeic acid formation. Addition of 5 mM p-coumaric acid into S. caeruleus cultures pre-grown on minimal medium with 1.0 g/l glucose resulted in the production of 65 mg/l caffeic acid. Furthermore, S. caeruleus cells were able to produce the maximum amount of caffeic acid when pre-grown on nutrient broth for 16 h. Under this condition, the addition of 5 mM p-coumaric acid was sufficient for the S. caeruleus culture to produce 150 mg/l caffeic acid, with a molar yield of 16.6% after 96 h of incubation.     

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.     

Dynamics of phenolic acids and lignin accumulation in metal-treated Matricaria chamomilla roots

Phenylalanine ammonia-lyase (PAL) activity, 11 phenolic acids and lignin accumulation in Matricaria chamomilla roots exposed to low (3 μM) and high (60 and 120 μM) levels of cadmium (Cd) or copper (Cu) for 7 days were investigated. Five derivatives of cinnamic acid (chlorogenic, p-coumaric, caffeic, ferulic and sinapic acids) and six derivatives of benzoic acid (protocatechuic, vanillic, syringic, p-hydroxybenzoic, salicylic acids and protocatechuic aldehyde) were detected. Accumulation of glycoside-bound phenolics (revealed by acid hydrolysis) was enhanced mainly towards the end of the experiment, being more expressive in Cu-treated roots. Interestingly, chlorogenic acid was extremely elevated by the highest Cu dose (21-fold higher than control) suggesting its involvement in antioxidative protection. All compounds, with the exception of chlorogenic acid, were detected in the cell wall bound fraction, but only benzoic acids were found in the ester-bound fraction (revealed by alkaline hydrolysis). Soluble phenolics were present in substantially higher amounts in Cu-treated roots and more Cu was retained there in comparison to Cd. Cu strongly elevated PAL activity (by 5.4- and 12.1-fold in 60 and 120 μM treatment, respectively) and lignin content (by 71 and 148%, respectively) after one day of treatment, indicating formation of a barrier against metal entrance. Cd had slighter effects, supporting its non-redox active properties. Taken together, different forms of phenolic metabolites play an important role in chamomile tolerance to metal excess and participate in active antioxidative protection.     

Metabolism of aromatic acids in the antibiotic-producing basidiomyceteOudemansiella mucida

Aromatic acids were determined in the mycelium and fermentation medium ofOudemansiella mucida. Coumaric acids (bothm- andp-),p-hydroxybenzoic acid (salicylic acid) and benzoic acid were found to predominate in the mycelium. Phenylacetic acid represents the main component in the medium. Phenylalanine ammonia-lyase catalyzing conversion of phenylalanine to cinnamie acid which is further metabolized to benzoic acid was detected in the mycelium. The results are discussed with respect to the synthesis of the antibiotic mucidin.     

Isolation and characterization of thermophilic bacilli degrading cinnamic, 4-coumaric,and ferulic acids

Thirty-four thermophilic Bacillus sp. strains were isolated from decayed wood bark and a hot spring water sample based on their ability to degrade vanillic acid under thermophilic conditions. It was found that these bacteria were able to degrade a wide range of aromatic acids such as cinnamic, 4-coumaric, 3-phenylpropionic, 3-(p-hydroxyphenyl)propionic, ferulic, benzoic, and 4-hydroxybenzoic acids. The metabolic pathways for the degradation of these aromatic acids at 60 degrees C were examined by using one of the isolates, strain B1. Benzoic and 4-hydroxybenzoic acids were detected as breakdown products from cinnamic and 4-coumaric acids, respectively. The beta-oxidative mechanism was proposed to be responsible for these conversions. The degradation of benzoic and 4-hydroxybenzoic acids was determined to proceed through catechol and gentisic acid, respectively, for their ring fission. It is likely that a non-beta-oxidative mechanism is the case in the ferulic acid catabolism, which involved 4-hydroxy-3-methoxyphenyl-beta-hydroxypropionic acid, vanillin, and vanillic acid as the intermediates. Other strains examined, which are V0, D1, E1, G2, ZI3, and H4, were found to have the same pathways as those of strain B1, except that strains V0, D1, and H4 had the ability to transform 3-hydroxybenzoic acid to gentisic acid, which strain B1 could not do.