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.     

Induction of phenylalanine ammonia-lyase activity by tryptophan in Ustilago maydis.

To understand the regulation of phenylalanine ammonia-lyase (PAL) activity in the corn smut fungus, Ustilago maydis, we examined the effects of different media, metabolic effectors (including aromatic amino acids), and environmental factors on induction and repression of PAL activity. PAL was detected only in cell extracts and not in the culture medium. U. maydis PAL is constitutively produced at a low level in all media tested but its regulation can be influenced by aromatic amino acids. L-Tryptophan (0.3 mM) induces PAL activity 3- to 5-fold but tryptophan analogs and tryptophan-related metabolites do not. The enzyme is most readily induced during the early stationary phase of growth and the induced activity remains relatively constant during stationary stage. No induction or inhibition of PAL activity was observed as a function of culture temperature, pH or light. PAL induction was repressed by glucose but not by its reaction product, t-cinnamic acid. Induction did not require de novo protein synthesis, suggesting that some form of post-translational protein modification or a metabolic effect may be involved. This study shows that the regulation of U. maydis PAL is very different from the patterns known for plants and other fungi.     

Phenolic synthesis and phenylalanine ammonia-lyase activity in suspension cultures of Acer pseudoplatanus L.

Summary Phenolic metabolism is influenced by the levels of sucrose, nitrogen and 2,4 dichlorophenoxyacetic acid (2,4-D) in the growth medium. Chromatographic evidence suggests that the principle products are polymers of leucocyanin, (-) epicatechin and (+) catechin, constituting condensed tannins. Comparison of ethanolic cell extracts with extracts from plant organs shows that although these compounds are present in parts of the plant they are not the major phenolics.Cells maintained in a modified Heller's medium containing 9.0×10^–7 M 2,4-D produce increased levels of tannins from mid passage (day 12) onwards. The presence of 2,4-D at 9.0×10^–6 M supresses this response and increased initial sucrose levels cause the amount of tannins to be greater. At the period when tannin levels increase the standard medium is exhaused of its nitrogen sources, urea and nitrate. Increased initial nitrogen levels delay the beginning of increased tannin production and the addition of urea or 2,4-D to cultures already containing high levels of tannins causes the tannin content per gram fresh weight and per culture to decline. These results indicate an antagonism between tannin synthesis and nitrogen metabolism. The activity of phenylalanine ammonia-lyase EC 4.1.1.5. (PAL) estimated by a spectrophotometric method in acetone powders derived from Acer cells increases three to four fold at the onset of increased tannin synthesis and then declines sharply. The phase of high PAL activity correlates with the exhausion of the medium nitrogen sources.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid One of the authors (R.J.W.) was supported by a Science Research Council Studentship during the course of this work.     

Further observations on phenylalanine ammonia-lyase in fungi

Phenylalanine ammonia-lyase (PAL) has been detected in an Ascomycete, Nectria cinnabarina. Growth in light increases levels of PAL in some but not all Basidiomycetes.     

Changes in phenylalanine ammonia-lyase activity in Satsuraa mandarin fruit during ontogeny

PAL activity in young fruits of Citrus unshiu was initiallyvery high. It decreased gradually as the fruit grew and afterthe "June drop", declined markedly. PAL behavior appears tobe closely related to the tissue differentiation of fruit. Proteincontent of the fruit showed a pattern of change similar to thatof PAL activity. Also, the amount of hesperidine was high inimmature fruit. Accumulation of hesperidine per fruit was greatlyenhanced as growth of the fruit progressed, but after PAL activityhad almost disappeared, the accumulation ceased. ¹Present address: Kozo Keikaku Engineering Incorporation, Nakano,Tokyo, Japan. (Received March 16, 1973; )     

Changes in phenylalanine ammonia-lyase activity during xylem differentiation in Coleus and soybean

Summary Xylem differentiation was induced in cultured Coleus internode slices when grown in the light on a simple agar/sucrose/IAA medium and in darkgrown soybean callus tissue when cultured on a complex defined medium containing 5×10^-7 M kinetin. In the Coleus system, the activity of phenylalanine ammonialyase followed the same time course as the formation of lignified wound vessel members. The specific activity of PAL was higher in the soybean callus tissues grown on 5×10^-7 M kinetin, which produced tracheary elements, than in the soybean tissue grown on 10^-8 M kinetin, which did not produce tracheids. These observations suggest that PAL is a marker enzyme for xylogenesis and that PAL activity may be a rate limiting step in lignification.Abbreviations IAA indole 3-yl acetic acid - NAA -naphthalene acetic acid - 2,4D 2,4,dichlorophenoxyacetic acid - DNA deoxyribose nucleic acid - TCA trichloracetic acid - PAL phenylalanine ammonia-lyase     

Elicitor-induced changes of phenylalanine ammonia-lyase activity in barley cell suspension cultures

Suspension-cultured barley cells responded to treatments with crude yeast extract and purified glucan preparation by rapidly and transiently (4 h postelicitation) inducing L-phenylalanine ammonia-lyase activity. Similarly, treatment of cell cultures with chitosan resulted in increased phenylalanine ammonia-lyase activity 2–4 h after elicitation, whereas a mycelium preparation of a fungal pathogen, Bipolaris sorokiniana, and purified chitin caused a more delayed induction of phenylalanine ammonia-lyase (8 h postelicitation). The most abundant of the plant cell wall degrading enzymes produced by Bipolaris sorokiniana, β-1,4-xylanase, had only a weak elicitor activity in barley cells suggesting that fungal cell wall components rather than the hydrolytic enzymes secreted by the fungus function as recognizable components that cause barley cells to induce defences. Treatment of the elicited cells with a phenylalanine ammonia-lyase inhibitor, α-aminooxy-β-phenylpropionic acid, resulted in the superinduction of the enzyme indicating the blocking of the feedback regulation mechanisms, whereas in the presence of 1 mM trans-cinnamic acid the elicitor-induction of phenylalanine ammonia-lyase was completely inhibited. Elicitor treatments increased the accumulation of wall-bound phenolics as evidenced by phloroglucinol-HCl staining and thioglycolic acid methods. However, α-aminooxy-β-phenylpropionic acid applied in combination with the elicitor did not prevent the accumulation of phenolics in barley cell walls. This suggested that phenylalanine ammonia-lyase might not play an important role in the synthesis wall-bound phenolic compounds in barley. However, cinnamic acid, whether applied alone or together with the elicitor, increased the amount of wall-bound phenolics in suspension-cultured barley cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Wound-induced phenylalanine ammonia-lyase in potato tuber tissue. Development of enzyme activity and effects of antibiotics.

Phenylalanine ammonia-lyase [EC 4.3.1.5.] activity increased rapidly after a 3-hr lag period in potato tuber (Solanum tuberosum L. cv. May Queen) disks incubated in a suitable medium in the dark at 25 degrees. The activity reached a maxinum after incubation for about 40 hr. The effects of actinomycin D, 6-methylpurine, cycloheximide, chloramphenicol, and mitomycin C on the induction of phenylalanine ammonia-lyase were investigated during incubation of the disks. Actinomycin D, 6-methylpurine, and cycloheximide all inhibited the formation of phenylalanine ammonia-lyase, though cycloheximide was the most effective at low concentrations. Application of actinomycin D for the initial lag period (3 hr) caused strong inhibition; however, if it was supplied later it did not inhibit but actually increased phenylalanine ammonialyase formation. In contrast, cycloheximide was effective over most of the incubation period. Chloramphenicol and mitomycin C did not inhibit phenylalanine phenylalanine ammonialyase induction, but markedly stimulated it. Light was not an essential factor for phenylalanine ammonia-lyase induction in the wounded tissue.     

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.     

Regulation of phenylalanine ammonia-lyase activity and growth in lettuce by light and gibberellic acid

Abstract The effects of light and gibberellic acid (GA3) on growth and phenylalanine ammonia-lyase (PAL) activity were studied in seedlings of lettuce (Lactuca sativa L.). Using an in vivo assay for PAL it was shown that wounding caused by excising hypocotyls results in an increase in PAL activity with time that can mask the effect of light on the activity of this enzyme. When hypocotyl sections were excised from light-treated seedlings immediately prior to the in vivo assay of PAL, light was shown to cause a marked increase in PAL activity. Experiments with an inhibitor of PAL activity, α-aminooxy-β-phenylpropionic acid (AOPP), confirmed that the volatile radioactive products measured in the in vivo assay resulted from the activity of PAL. Gibberellic acid suppresses the light-induced increase in PAL activity and there is an inverse relationship between GA₃-induced growth and the activity of PAL. Over a wide range of GA₃ concentrations, the activity of PAL is also inversely correlated with growth rate along the length of the hypocotyl section; the upper halves of sections elongate more rapidly and have lower levels of PAL than the lower halves. Despite the strong correlation between growth and PAL activity, experiments with AOPP and t-cinnamic acid show that it is unlikely that elongation is regulated directly by products of PAL activity.     

Synthesis and degradation of phenylalanine ammonia-lyase of Rhodosporidium toruloides.

The regulation of the enzyme phenylalanine ammonia-lyase (PAL), which is of potential use in oral treatment of phenylketonuria, was investigated. Antiserum against PAL was prepared and was shown to be monospecific for the enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme and two inactive mutant forms of the enzyme were purified to homogeneity by immunoaffinity chromatography, using anti-PAL immunoglobulin G-Sepharose 4B. Both mutant enzymes contained intact prosthetic groups. The formation of PAL catalytic activity after phenylalanine was added to yeast cultures was paralleled by the appearance of enzyme antigen. During induction, uptake of [3H]leucine into the enzyme was higher than uptake into total protein. Our results are consistent with de novo synthesis of an enzyme induced by phenylalanine, rather than activation of a proenzyme. The half-lives of PAL and total protein were similar in both exponential and stationary phase cultures. No metabolite tested affected the rate of enzyme degradation. Glucose repressed enzyme synthesis, whereas ammonia reduced phenylalanine uptake and pool size and so may repress enzyme synthesis through inducer exclusion. The synthesis of enzyme antigen by a mutant unable to metabolize phenylalanine indicated that this amino acid is the physiological inducer of the enzyme.     

Purification and properties of phenylalanine ammonia-lyase in cut-injured sweet potato.

L-Phenylalanine ammonia-lyase (PAL) activity was developed in response to cut injury in sweet potato root tissue. The enzyme was purified from tissue incubated for 1 day after slicing by ammonium sulfate fractionation, column chromatographies on L-phenylalanyl Sepharose 4B, phosphocellulose. Sephadex G-200 and Sepharose 6B and preparative polyacrylamide gel electrophoresis. The molecular weight and sedimentation coefficient were estimated to be 285,000 to 320,000 and 11.6 to 11.9 S, respectively. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gel yielded a single stained protein band which corresponded to a subunit weight of 80,000. Thus, the enzyme seems to be composed of four subunits of the same size. Neither L-tyrosine nor D-phenylalanine served as a substrate. Two Km values for the PAL were observed above and below 30 micrometers at various temperatures and were lower than those for PALs of other plants. The slope of the Arrhenius plot had a discontinuity at 17 degrees C. The values of activation energy were calculated to be 15,000 cal and 19,000 cal above and below 17 degrees C, respectively. Similar discontinuities were also observed in the effect of temperature on the Km values and the Hill coefficients. Negative cooperativity was observed at 10 degrees C (n = 0.83), but was not marked above 20 degrees C (n = 0.94).     

Chitosan and chitin oligomers increase phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities in soybean leaves

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and tyrosine ammonia-lyase (TAL, 4.3.1.), the key enzymes of the phenylpropanoid pathway, are inducible in response to biotic (such as chitin from fungal cell walls) and abiotic cues. Application of chitin and chitosan to soybean leaf tissues caused increased activity of PAL and TAL enzymes. The elevation of enzyme activity was dependent on the chain length of the oligomers and time after treatment. The hexamer of chitin and pentamer of chitosan produced the maximum activities at 36 h after treatment as compared to controls. Total phenolic content of soybean leaves increased following chitosan and chitin oligomer treatments, showing a positive correlation between enzyme activity and total phenolic content.     

Identification by high-performance liquid chromatography of tyrosine ammonia-lyase activity in purified fractions of Phaseolus vulgaris phenylalanine ammonia-lyase

Activities of phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) were assessed at each stage of a three-step purification of PAL. Assays were performed by high-performance liquid chromatographic (HPLC) separation and ultraviolet detection of reaction products. Use of HPLC permitted assay of low activities of PAL and TAL for periods up to approximately four and two days, respectively. HPLC also facilitated the accurate quantitation of the product of the TAL reaction, trans-p-coumaric acid, which was observed to isomerize readily under experimental conditions. PAL and TAL were associated throughout the purification procedure, with TAL activity at 0.6–1.3% of PAL activity. It was concluded that, contrary to previous reports, TAL and PAL activities are mediated by the same enzyme, or else by chromatographically very similar enzymes.     

Effect of glycerol,polyethylene glycol and glutaraldehyde on stability of phenylalanine ammonia-lyase activity in yeast

Summary The polyhydric alcohols, glycerol and sorbitol, significantly increased the rate ofl-phenylalanine production from trans-cinnamic acid using whole cells ofRhodotorula rubra. Chloride ions and oxygen prevented the stimulatory effect of the polyhydric alcohols. Furthermore, the severe inhibition, of the biotransformation by high trans-cinnamic acid concentrations was alleviated in the presence of glycerol, and sorbitol. The rate of conversion could be manipulated still further, even with high trnas-cinnamic acid concentrations, by elevating the reaction pH to, 12 in the presence of polyhydric alcohol. When cells were also treated first with glutaradehyde (0.1% v/v) and then polyethylene glycol (15% v/v), although neither compound stimulated the actual rate of bioconversion, the reaction was markedly stabilised and gave a 73% yield after 28 days of continuous operation.     

The effect of herbicides,plant growth regulators and other compounds on phenylalanine ammonia-lyase activity

The in vitro and in vivo effects of a number of herbicides and plant growth regulators on phenylalanine ammonia-lyase (PAL) activity were investigated. The most elective in vitro inhibitors were product analogs, t-cinnamic and p-coumaric acids, and carbonyl reagents, hydroxylamine and nitromethane. Application of the herbicides diuron, dalapon, amiben, and chloropropham, to plants resulted in a decrease in the intracellular concn of PAL. The inhibitory effect of alachlor was found to be dose-responsive and somewhat specific. A correlation between PAL inhibition and herbicidal activity was observed for hydroxylamine. The cytokinin, pyranyl benzyladenine, (PBA) increased PAL activity in pigweed. The possibility of developing herbicides acting through PAL inhibition is discussed.