Circadian Rhythmicity in the Activities of Phenylalanine Ammonia-Lyase from Lemna perpusilla and Spirodela polyrhiza

The oscillations in phenylalanine ammonia-lyase activity from Spirodela polyrhiza and phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities from Lemna perpusilla displayed a circadian rhythm under continuous light. Rhythmicity in enzymic activity could not be detected in continuous darkness since under this condition phenylalanine ammonia-lyase activity remains at a fairly constantly low level. Results from our studies of the oscillatory pattern of the respective activities of phenylalanine and tyrosine ammonia-lyase support their “inseparability.”     

Phenylalanine and tyrosine ammonia-lyase activity in Sporobolomyces pararoseus

Phenylalanine ammonia-lyase from Sporobolomyces pararoseus was purified more than 450-fold. Polyacrylamide disc gel electrophoresis of this purified enzyme gave a single major protein band. Tyrosine ammonia-lyase activity was monitored during the purification of phenylalanine ammonia-lyase. Deaminating activities for phenylalanine and tyrosine were not separated during the purification process. The existence of one ammonia-lyase with bisubstrate activity is postulated.     

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.     

The Site of the Inhibition of the Shikimate Pathway by Glyphosate: I. INHIBITION BY GLYPHOSATE OF PHENYLPROPANOID SYNTHESIS IN BUCKWHEAT (FAGOPYRUM ESCULENTUM MOENCH)

The nonselective herbicide glyphosate (n-[phosphonomethyl]glycine) inhibited the light-induced accumulation of phenylpropanoid substances (chlorogenic acid, procyanidin, rutin, anthocyanin) in etiolated buckwheat hypocotyls 90% at 1 millimolar. Structurally related compounds, such as n,n-bis[phosphonomethyl]glycine, aminomethylphosphonate, methylglycine, and iminodiacetate, had little or no inhibiting effects. Of all amino acids tested, only l-phenylalanine reversed the inhibition, and partial reversal of anthocyanin synthesis was achieved with chorismate, phenylpyruvate, trans-cinnamate, p-coumarate, and naringenin. Phenylalanine concentrations were reduced in glyphosate-treated hypocotyls, and glyphosate effectively reduced the high level of phenylalanine that was caused by the phenylalanine ammonia-lyase inhibitor l-alpha-aminooxy-beta-phenylpropionate. Glyphosate had no significant effect on the time course of phenylalanine ammonia-lyase activity in hypocotyls incubated either in the dark or in the light. Under appropriate feeding conditions, glyphosate inhibited the incorporation of [(14)C]shikimate into all three aromatic amino acids, and radioactive shikimate accumulated in the tissue. The results lead to the conclusion that glyphosate interferes with the shikimate pathway at or prior to the formation of chorismate.     

On the Mechanism of the Changes in Phenylalanine Ammonia-lyase Activity Induced by Ultraviolet and Blue Light in Gherkin Hypocotyls

Irradiation with ultraviolet light causes in the hypocotyl of dark-grown gherkin seedlings the partial conversion of trans-hydroxycinnamic acids to the cis-isomers. The trans-hydroxycinnamic acids inhibit the development of phenylalanine ammonia-lyase activity, and the transformation of these compounds to the much less inhibitory cis-isomers forms a ready explanation for the increase in phenylalanine ammonia-lyase activity in the hypocotyl of gherkin seedlings irradiated with ultraviolet light. Arguments are advanced that the increase in phenylalanine ammonia-lyase activity caused by irradiation with blue light is also (at least in part) initiated by trans-cis isomerisation of the hydroxycinnamic acids.     

Entrapment of phenylalanine ammonia-lyase in silk fibroin for protection from proteolytic attack

Phenylalanine ammonia-lyase was entrapped in silk fibroin. The entrapped enzyme showed a similar Km for Phe and pH optimum to the free enzyme. It was resistant against chymotrypsin and trypsin in vitro. To assess the activity in vivo, the free or entrapped enzymes and then Phe were injected into rat duodenum, and cinnamate, a product, in plasma was determined as the most direct evidence of the enzyme activity. The entrapped enzyme but not the free form caused a marked raise of plasma cinnamate. It declined with a half life of about 45 min, which was significantly longer than that (10-15 min) observed upon i.v. administration of cinnamate. These results indicated that the entrapped enzyme was actively degrading Phe in the intestinal tract. Entrapment of phenylalanine ammonia-lyase in fibroin thus provides a new prospect for oral enzyme therapy of phenylketonuria.     

Reconstitution of the entry point of plant phenylpropanoid metabolism in yeast (Saccharomyces cerevisiae): implications for control of metabolic flux into the phenylpropanoid pathway

Phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and the C4H redox partner cytochrome p450 reductase (CPR) are important in allocating significant amounts of carbon from phenylalanine into phenylpropanoid biosynthesis in plants. It has been proposed that multienzyme complexes (MECs) containing PAL and C4H are functionally important at this entry point into phenylpropanoid metabolism. To evaluate the MEC model, two poplar PAL isoforms presumed to be involved in either flavonoid (PAL2) or in lignin biosynthesis (PAL4) were independently expressed together with C4H and CPR in Saccharomyces cerevisiae, creating two yeast strains expressing either PAL2, C4H and CPR or PAL4, C4H and CPR. When [(3)H]Phe was fed, the majority of metabolized [(3)H]Phe was incorporated into p-[(3)H]coumarate, and Phe metabolism was highly reduced by inhibiting C4H activity. PAL alone expressers metabolized very little phenylalanine into cinnamic acid. To test for intermediate channeling between PAL and C4H, we fed [(3)H]Phe and [(14)C]cinnamate simultaneously to the triple expressers, but found no evidence for channeling of the endogenously synthesized [(3)H]cinnamate into p-coumarate. Therefore, efficient carbon flux from Phe to p-coumarate via reactions catalyzed by PAL and C4H does not appear to require channeling through a MEC in yeast, and instead biochemical coupling of PAL and C4H is sufficient to drive carbon flux into the phenylpropanoid pathway. This may be the primary mechanism by which carbon allocation into phenylpropanoid metabolism is controlled in plants.     

Maize phenylalanine ammonia-lyase has tyrosine ammonia-lyase activity.

A full-length cDNA encoding phenylalanine ammonia-lyase (PAL) from Zea mays L. was isolated and the coding region was expressed in Escherichia coli as a C-terminal fusion to glutathione S-transferase. After purification by glutathione-Sepharose chromatography, the glutathione S-transferase moiety was cleaved off and the resulting PAL enzyme analyzed. In contrast to PAL from dicots, this maize PAL isozyme catalyzed the deamination of both L-phenylalanine (PAL activity) and L-tyrosine (tyrosine ammonia-lyase activity). These results provide unequivocal proof that PAL and tyrosine ammonia-lyase activities reside in the same polypeptide. In spite of large differences in the Michaelis constant and turnover number of the two activities, their catalytic efficiencies are very similar. Also, both activities have the same pH and temperature optima. These results imply that maize can produce p-coumaric acid from both phenylalanine and tyrosine.     

2. Alicyclic acid metabolism in plants Enzymes related to the shikimate pathway in developing mung bean seedlings

A study was made of chages in the activities of enzymes relatedto the biosynthesis of aromatic compounds in etiolated mungbean seedlings during their growth. Shikimate: NADP oxidoreductaseactivity in the root-shoot axes increased rapidly to attainits highest activity the 4th day after sowing, and remainedat that level over the experimental period of 7 days. 5-Dehydroquinatehydro-lyase activity continuously increased for at least 7 days.In the cotyledons, a gradual decrease in the activities of theseenzymes occurred. Phenylalanine ammonia-lyase activity in root-shootaxes gradually increased showing a maximum on the 6th day. Thehighest specific activity, on a protein basis, of this enzymewas seen in the initial stage of growth. In the cotyledons,a rise in total activity appeared on the 2nd day. Tyrosine ammonia-lyaseactivity was very low as compared with phenylalanine ammonia-lyase.The enzyme activities of light-germinated seedlings were comparedto those of dark-germinated seedlings on the 7th day. Lighthad practically no significant effect on the appearance of shikimate:NADP oxidoreductase and 5-dehydroquinate hydro-lyase activities.On the other hand, a marked effect from the light on the riseof phenylalanine ammonia-lyase and tyrosine ammonia-lyase activitieswas found, especially in the epicotyl-plumules. The results are discussed with respect to the metabolism ofalicyclic acids such as shikimic acid in the developing mungbean seedlings. ¹This work was partly supported by a grant-in-aid from the Ministryof Education.     

Activities of Phenylalanine- and Tyrosine-Ammonia Lyases and Aminotransferases during Organogenesis in Callus Cultures of Rice

Activities of key enzymes involved in the shikimic acid pathway,namely, phenylalanine ammonia-lyase, tyrosine ammonia-lyase,phenylalanine aminotransferase and tyrosine aminotransferase,were examined during initiation of roots and shoots in calluscultures of rice. Enhancement of the activities of tyrosine-ammonialyase and tyrosine aminotransferase but not of phenylalanine-ammonialyase and phenylalanine aminotransferase was observed in organ-formingcultures, as compared to activities of these enzymes in non-organ-formingcultures. The role of these enzymes in such organogenetic processesis discussed (Received March 16, 1988; Accepted October 19, 1988)     

Rosmarinic acid formation and differential expression of tyrosine aminotransferase isoforms in Anchusa officinalis cell suspension cultures

Time-course changes in rosmarinic acid (RA) formation and activities of tyrosine aminotransferase (TAT) isoforms were examined in Anchusa officinalis suspension cultures. Three TAT isoforms (TAT-1, TAT-3, TAT-4) were resolved by Mono-Q anion-exchange column chromatography. The proportion of the TAT-3 activity within the total TAT activity remained high regardless of the growth stage of the cultured cells. TAT-1 activity was positively correlated with the rate of RA biosynthesis during linear growth stage of the culture cycle, while TAT-4 activity was rapidly induced in conjunction with transfer to fresh medium coincident with a transient increase in RA synthesis. Based on these results, as well as the substrate specificity of each TAT isoform, it was concluded that both TAT-1 and TAT-4 are closely involved in RA biosynthesis. TAT-1 controls conversion of tyrosine to 4-hydroxyphenyl pyruvate, and TAT-4 acts by participating in the formation of tyrosine and phenylalanine via prephenate.Abbreviations PAL phenylalanine ammonia-lyase - TAT tyrosine aminotransferase - RA rosmarinic acid     

Possible errors in quantitative determination of phenylalanine ammonia-lyase activity by spectrophotometric methods

A possible error in spectrophotometric determination of cinnamate, the product of phenylalanine ammonia-lyase activity, using nonpurified protein extracts has been shown.     

Molecular regulation of catechins biosynthesis in tea [Camellia sinensis (L.) O. Kuntze

Catechins are bioprospecting molecules present in tea and any effort towards metabolic engineering of this important moiety would require knowledge on gene regulation. These are synthesized through the activities of phenylpropanoid and flavonoid pathways. Expression regulation of various genes of these pathways namely phenylalanine ammonia-lyase (CsPAL), cinnamate 4-hydroxylase (CsC4H), p-coumarate:CoA ligase (Cs4CL), flavanone 3-hydroxylase (CsF3H), dihydroflavonol 4-reductase (CsDFR) and anthocyanidin reductase (CsANR) was accomplished previously. In depth analyses of the remaining genes namely, chalcone synthase (CsCHS), chalcone isomerase (CsCHI), flavonoid 3'5'-hydroxylase (CsF3'5'H) and anthocyanidin synthase (CsANS) were lacking. The objective of the work was to clone and analyze these genes so as to generate a comprehensive knowledge on the critical genes of catechins biosynthesis pathway. Gene expression analysis was carried out in response to leaf age and external cues (drought stress, abscisic acid, gibberellic acid treatments and wounding). A holistic analysis suggested that CsCHI, CsF3H, CsDFR, CsANS and CsANR were amongst the critical regulatory genes in regulating catechins content.     

Induction and characterization of a microsomal flavonoid 3'-hydroxylase from parsley cell cultures

A microsomal preparation from irradiated parsley cell cultures catalyses the NADPH and dioxygen-dependent hydroxylation of (S)-naringenin [(S)-5, 7, 4'-trihydroxyflavanone] to eriodictyol (5, 7, 3', 4'-tetrahydroxyflavanone). Dihydrokaempferol, kaempferol, and apigenin were also substrates for the 3'-hydroxylase reaction. In contrast prunin (naringenin 7-O-beta-glucoside) was not converted by the enzyme. The microsomal preparation, which also contains cinnamate 4-hydroxylase, did not catalyse hydroxylation of 4-coumaric acid to caffeic acid. 3'-Hydroxylase activity is partially inhibited by carbon monoxide in the presence of oxygen as well as by cytochrome c and NADP+. These properties suggest that the enzyme is a cytochrome P-450-dependent flavonoid 3'-monooxygenase. Pronounced differences in the inhibition of flavonoid 3'-hydroxylase and cinnamate 4-hydroxylase were found with EDTA, potassium cyanide and N-ethylmaleimide. Irradiation of the cell cultures led to increase of flavonoid 3'-hydroxylase activity with a maximum at about 23 h after onset of irradiation and subsequent decrease. This is similar to light-induction of phenylalanine ammonialyase and cinnamate 4-hydroxylase. In contrast, treatment of the cell cultures with a glucan elicitor from Phytophthora megasperma f. sp. glycinea did not induce flavonoid 3'-hydroxylase nor chalcone isomerase but caused a strong increase in the activities of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, and NADPH--cytochrome reductase. The results prove that flavonoid 3'-hydroxylase and cinnamate 4-hydroxylase are two different microsomal monooxygenases.     

Phytochrome-induced flavonoid biosynthesis in mustard (Sinapis alba L.) cotyledons. Enzymic control and differential regulation of anthocyanin and quercetin formation

Phytochrome-induced increases in enzyme activities for phenylalanine ammonia-lyase (EC 4.3.1.5) and chalcone isomerase (EC 5.5.1.6), and in amounts of the related end products, anthocyanin and the flavonol, quercetin, were measured in cotyledons of mustard (Sinapis alba L.). There was no correlation between the activities of these enzymes and the rate of anthocyanin accumulation; however, some correlation was found with the quercetin accumulation rate. Since anthocyanin and flavonol accumulation is spatially separated in mustard (flavonols in the upper epidermis, anthocyanin in the lower epidermis), it was possible to measure anthocyanin-associated phenylalanine ammonia-lyase independently. This activity correlated well with the accumulation rate for anthocyanin during the first few hours after induction. The phytochrome effect on anthocyanin formation differed from that on quercetin formation: anthocyanin was strongly induced by continuous far-red light and by both continuous red light and red light pulses, whereas quercetin was only effectively induced by continuous far-red light.Abbreviations CHI chalcone isomerase - PAL phenylalanine ammonia-lyase     

Induction of biosynthetic enzymes for avenanthramides in elicitor-treated oat leaves

The accumulation of oat (Avena sativa L.) phytoalexins, avenanthramides, occurred in leaf segments treated with oligo-N-acetylchitooligosaccharides. The amount of avenanthramide A, the major oat phytoalexin, reached a maximum 36–48 h after elicitor treatment. This accumulation was preceded by a marked increase in enzyme activities of phenylpropanoid pathway members, including phenylalanine ammonia-lyase (EC 4.3.1.5), cinnamate 4-hydroxylase (EC 1.14.13.11) and 4-coumarate:CoA ligase (EC 6.2.1.12). These enzyme activities reached a maximum 6–12 h after elicitor treatment, when the avenanthramides were produced most rapidly. Both phenylalanine ammonia-lyase and 4-coumarate:CoA ligase activities decreased thereafter to undetectable levels 72 h after treatment, while cinnamate 4-hydroxylase activity showed a second increase 48 h after treatment. Among the chitooligosaccharides tested, tetra- and pentasaccharides most effectively induced these enzyme activities in a dose-dependent manner. The elicitor-induced 4-coumarate: CoA ligase accepted all hydroxycinnamic acids occurring in the avenanthramides as substrates, with the exception of avenalumic acid. These findings indicate that accumulation of the avenanthramides results from de-novo synthesis through the general phenylpropanoid pathway and that early biosynthetic enzymes function as regulatory points of carbon flow to the avenanthramides. Received: 3 December 1998 / Accepted: 27 January 1999     

Recherches sur les enzymes catalysant la biosynthèse des acides phénoliques chez Quercus pedunculata. III. Formation séquentielle, à partir de la phénylalanine, des acides cinnamique, p-coumarique et caféique, par des organites cellulaires isolés

The reactions leading to cinnamic acids from phenylalanine as only substrate were investigated in organelles from Quercus pedunculata Ehrh. roots. –“F 10 000′” fraction, including mitochondria and micro-bodies, catalyses the first reaction, i.e., cinnamate formation by deamination of phenylalanine. – Microsomal fraction catalyses all the steps from phenylalanine to caffeic acid via cinnamate and p-coumarate. These results suggest that microsomes are the intracellular site of the cinnamic units synthesis. The enzymes involved in these reactions, associated in the same cellular compartment, does not form a multienzyme system. The formation of caffeic acid by isolated microsomes is demonstrated for the first time; the reaction may be realised by an enzyme different from phenolase. – The free phenolic acids are the metabolically active forms.     

Methyl jasmonate-induced rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures

Summary A dramatic increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after their exposure to methyl jasmonate (MJ). Preceding the induced RA accumulation, phenylalanine ammonia-lyase (PAL) and 4-hydroxyphenylpyruvate reductase (HPR) activities increased rapidly and transiently, whereas tyrosine aminotransferase (TAT) activity showed only a slight increase. The elicitation activity of MJ was much higher than that of yeast extract (YE) in terms of the induction of PAL and HPR activities, RA accumulation and incorporation of both ¹⁴C-phenylalanine and ¹⁴C-tyrosine into RA. However, the response of the cultured cells to MJ-treatment was slower than that to YE-treatment.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - LS Linsmaier and Skoog - HPR 4-hydroxyphenylpyruvate reductase - PAL phenylalanine ammonia-lyase - TAT tyrosine aminotransferase - MJ methyl jasmonate - YE yeast extract     

Differential response of cultured parsley cells to elicitors from two non-pathogenic strains of fungi. 2. Effects on enzyme activities

Parsley cell cultures produce linear furanocoumarins and the linear benzodipyrandione, graveolone, in response to treatment with an elicitor from either Phytophthora megasperma or Alternaria carthami. Activities of enzymes involved in general phenylpropanoid metabolism, phenylalanine ammonia-lyase and 4-coumarate: CoA ligase, as well as of an enzyme involved specifically in furanocoumarin biosynthesis, dimethylallyl diphosphate: umbelliferone dimethylallyltransferase, were monitored over several days after treatment with A. carthami elicitor. In addition, the activities of chalcone synthase, an enzyme involved in flavonoid formation, and of glucose-6-phosphate: NADP 1-oxidoreductase were also monitored. The lyase and the ligase activities increased steadily for 48 h and the dimethylallyltransferase activity for 54 h, while the synthase activity was not altered and the oxidoreductase activity decreased gradually. In some experiments, phenylalanine ammonia-lyase activity reached a maximum value of 250 mukat/kg, twice the maximal activity observed previously in parsley cells after treatment with either ultraviolet light or an elicitor preparation from P. megasperma. In crude extracts, phenylalanine ammonia-lyase activity was shown to be inhibited by unidentified small-molecular-weight compounds which were formed in proportion to the elicitor treatment. While phenylalanine ammonia-lyase and dimethylallyl diphosphate: umbelliferone dimethylallyltransferase are known to be required for furanocoumarin biosynthesis, the involvement of 4-coumarate: CoA ligase is as yet unclear. The concomitant increase and decrease of the ligase activity with the activities of the lyase and the dimethylallyltransferase, as well as its similar response to elicitor concentrations, suggest that CoA esters of cinnamic acids play a role in the biosynthesis of furanocoumarins.