Ammonia Lyase Research Articles

Organ‐ and Tissue‐specific Biosynthesis of Flavonoids in Seedlings of Oenothera odorata (Onagraceae)

AbstractIn developing Oenothera odorata seedlings, phytochrome‐mediated accumulation of various flavonoids (mainly glycosides of cyanidin and quercetin) is detectable, subsequent to a transient induction of the key enzymes of the general phenylpropanoid metabolism, L‐phenylalanine ammonia lyase (PAL) and of flavonoid biosynthesis, chalcone synthase (CHS). Organ‐ and tissue‐specific distribution of these enzymes and of the flavonoid end products was investigated in seedlings, irradiated with continuous far‐red light.Anthocyanins and quercetin glycosides are mainly localized in both the upper and lower epidermis of the cotyledons and to a lesser extent also in the epidermal cell layer of the hypocotyl. An obvious organ‐specific distribution was observed for the anthocyanins: cyanidin‐3,5‐O‐diglucoside accumulates in the epidermal cells of the cotyledons, whereas cyanidin‐3‐O‐glucoside is restricted to the epidermis of the hypocotyl. By contrast the pattern of quercetin glycosides is the same in the cotyledons and in the hypocotyl. The methylated flavonol aglycone 3‐0‐methylquercetin was found to be localized in the seed coat. According to this organ‐ and tissue‐specific pattern of flavonoids, immunochemical and immunohistochemical detection of PAL and CHS revealed a predominant localization of theenzymes in the epidermal layers of the cotyledons and the hypocotyl but also in the cells surrounding the vascular bundles. The role of compartmentation in regulation of flavonoid biosynthesis and putative functions of flavonoid compounds are discussed.

REGULATION OF UV‐PROTECTIVE PIGMENT SYNTHESIS IN THE EIPERMAL LAYER OF RYE SEEDLINGS (Secale cereale L. cv. KUSTRO)

Abstract Trans‐cinnamic acid is the product of the L‐phenylalanine ammonia lyase (PAL, EC 4.3.1.5) enzyme reaction and might regulate the subsequent phenylpropanoid synthesis by feedback inhibition of PAL activity. The partial conversion of trans‐cinnamic acids (e.g. trans‐p‐coumaric acid) and of trans‐p‐coumaryl‐glucoside (chemically synthesized model substance for cinnamic acid derivatives) to the cis isomers upon UV irradiation leads to reduced inhibition of PAL activity within minutes. In rye leaves this rapid enhancement of PAL activity (within minutes) causes a rapid increase in phenylpropanoids, which are predominantly located in the epidermal layer. Because phenylpropanoid derivatives are precursors of flavonoid biosynthesis, the observed rapid increase in flavonoid content after short‐term UV irradiation can be explained by this UV‐induced regulation mechanism. Consequently, transphenylpropanoids can be regarded as photoreceptors and regulators of the phenylpropanoid pathway during UV‐B (280–320nm) irradiation. In rye leaves an additional slower UV response, obviously by induction of genetic transcription as described for other plant systems, leads to a much greater flavonoid accumulation compared to the rapid induction.

Rapid Activation of Phenylpropanoid Metabolism in Elicitor-Treated Hybrid Poplar (Populus trichocarpa Torr. & Gray × Populus deltoides Marsh) Suspension-Cultured Cells

Elicitor induction of phenylpropanoid metabolism was investigated in suspension-cultured cells of the fast-growing poplar hybrid (Populus trichocarpa Torr. & Gray x Populus deltoides Marsh) H11-11. Treatment of cells with polygalacturonic acid lyase or two fungal elicitors resulted in rapid and transient increases in extractable l-phenylalanine ammonia lyase and 4-coumarate:coenzyme A ligase enzyme activities. The substrate specificity of the inducible 4-coumarate:coenzyme A ligase enzyme activity appeared to differ from substrate specificity of 4-coumarate:coenzyme A ligase enzyme activity in untreated control cells. Large and transient increases in the accumulation of l-phenylalanine ammonia-lyase and 4-coumarate:coenzyme A ligase mRNAs preceded the increases in enzyme activities and were detectable by 30 minutes after the start of elicitor treatment. Chalcone synthase, cinnamyl alcohol dehydrogenase, and coniferin beta-glucosidase enzyme activities were unaffected by the elicitors, but a large and transient increase in beta-glucosidase activity capable of hydrolyzing 4-nitrophenyl-beta-glucoside was observed. Subsequent to increases in l-phenylalanine ammonialyase and 4-coumarate:coenzyme A ligase enzyme activities, cell wall-bound thioglycolic acid-extractable compounds accumulated in elicitor-treated cultures, and these cells exhibited strong staining with phloroglucinol, suggesting the accumulation of wall-bound phenolic compounds.

ChemInform Abstract: Stereochemical Course of the Enzymic Amination of Chloro‐ and Bromofumaric Acid by 3‐Methylaspartate Ammonia‐Lyase.

AbstractThe halofumarates (I) are aminated in the presence of 3‐methylaspartate ammonia lyase, yielding the betaines (II).

Phenolic Materials in Auxin Heterotroph and Autotroph (Habituated) Tobacco Callus Tissues

The total phenolic content and activity of phenylalanine ammonium lyase in auxin autotroph (habituated) and heterotroph tobacco callus were compared, and no significant difference was indicated.The elongation of Avena seedling segments was inhibited more significantly by extracted phenolics of heterotroph than autotroph callus.p-Coumaric and ferulic acid were found to accumulate preferentially in heterotroph callus tissue.The multiple effect of these two phenols on fresh weight and in vitro PAL activity of tobacco calli is investigated and discussed.

Metabolism of 2,3-diaminopropionate in the rat

L-[U-14C]-2, 3-diaminopropionate was metabolised slowly in the rat. Nearly 75% of the total radioactivity could be accounted for by respiratory CO2 (60%) and by the concentration of the isotope in the liver and kidney (15%). The rate limiting step in its metabolism may be the activity of 2, 3-diaminopropionate: ammonia lyase in the liver. It is more readily metabolised than its 3-oxalyl derivative, which is neurotoxic.

Phenylalanine ammonia lyase and cinnamic acid hydroxylases as assembled consecutive enzymes on microsomal membranes of cucumber cotyledons: Cooperation and subcellular distribution

1. Cooperation between phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and cinnamic acid hydroxylases was investigated using microsomal fractions from cotyledons of cucumber (Cucumis sativus L.). The interpretations were based on experiments which demonstrate a limited exchange between the pool of cinnamic acid formed by the membrane-bound phenylalanine ammonia-lyase and the cinnamic acid pool external to the enzyme-membrane system. 2. The extent of cooperation between the microsomal enzymes was proved to be influenced by treatment of the cotyledons with light. On exposure to UV-light, which is known to enhance greatly the soluble phenylalanine ammonia-lyase activity in cell cultures, differential effects on the levels of microsomal and soluble phenylalanine ammonia-lyase, and of cinnamic acid hydroxylases, were observed. The time course of the enzyme activities and their cooperation in vitro after treatment of the cotyledons with light were studied. 3. The extent of cooperation in vitro was found to vary depending on the concentration of L-phenylalanine. 4. Homogenates obtained from etiolated cotyledons of Cucumis sativus in the absence of Mg(2+) were fractionated by sucrose density gradient centrifugation and examined for phenylalanine ammonia-lyase, cinnamic acid o-hydroxylase, cinnamic acid o-hydroxylase, and several marker enzymes. Ammonia-lyase activity was highest in fractions with 25% sucrose, in which primarily smooth endoplasmic reticulum is localized. Hydroxylase activities co-occur with phenylalanine ammonia-lyase in these fractions (density=1.100 g/cm(3)), and also in fractions at higher densities (d=1.12-1.13 and 1.15 g/cm(3)).

Density-labelling evidence for the blue-light-mediated activation of phenylalanine ammonia lyase in Cucumis sativus seedlings

Evidence is presented which demonstrates that both acid phosphatase (EC 3.1.3.2) and phenylalanine ammonia-lyase (EC 4.3.1.5) are synthesised in the hypocotyls of dark-grown gherkin seedlings. When blue light or cycloheximide treatment is given in the presence of 2H 2 O the buoyant density of the lyase is observed to be lower than the appropriate dark control. This effect is not found for the phosphatase, the buoyant density of which is unaffected by blue ligh. These data appear to be best interpreted as a blue-light- and cycloheximide-mediated activation of previously synthesised, inactivated phenylalanine ammonia-lyase.

Phenylalanine ammonia lyase, chlorogenic acid and lignin in potato tuber tissue inoculated with Phytophthora infestans

Tuber disks of Majestic (susceptible-r) and Orion (resistant-R 1) were inoculated with a sporangial suspension of Phytophthora infestans race 4. There were increases in the activity of phenylalanine ammonia-lyase and the level of chlorogenic acid, accompanied by the accumulation of a lignin-like polymer. All three changes were faster in Orion than in Majestic. Lignification was relatively slow and diffuse in the widespread infected area of slices from Majestic but more rapid in the centre of the restricted infected area of Orion slices. Phenylalanine ammonia-lyase activity followed a similar pattern. It seems probable that lignification is involved in the restriction of the spread of the pathogen in Orion slices, and that it is controlled in part by the level of phenylalanine ammonia-lyase.

Studies on the identification and characterization of an aspartase activity in liver of elasmobranch fishes

1. 1. l-aspartate is actively deaminated by elasmobranch fish liver: experiments with 15N-aspartate demonstrate the formation of 15N-ammonia. 2. 2. The mechanism of the amino group detachment from l-aspartate has been investigated. Fumarate formation in stoichiometric amounts with ammonia, and experiments performed in the presence of α-oxoglutarate, l-glutamate, NAD.† Inosinic acid and other compounds support a direct mechanism of aspartate deamination rather than an indirect one. The enzyme activity has been visualized as an aspartate : ammonia lyase (aspartase), never found before in animal tissues. 3. 3. The enzyme activity has been characterized, it has been found moderately stable and is associated with a homogeneous mitochondrial fraction. Attempts at its solubilization from the mitochondrial matrix with detergents, ultrasounds, etc. were unsuccessful. 4. 4. The biological significance of this enzyme activity in elasmobranch fishes is discussed.

The Metabolism of O-Phosphorylethanolamine in Animal Tissues: I. O-PHOSPHORYLETHANOLAMINE PHOSPHO-LYASE: PARTIAL PURIFICATION AND CHARACTERIZATION

Abstract The enzyme O-phosphorylethanolamine phospho-lyase has been purified 107-fold from rabbit liver and 580-fold from rat liver with the use of DEAE-cellulose and brushite chromatography. The pH optimum of the enzyme's activity is 7.8 and enzyme preparations are unstable at temperatures above 55°. The apparent Km for O-phosphorylethanolamine was found to be 6.1 x 10-4. Dialyzed preparations after treatment with hydroxylamine were found to be almost completely dependent on the addition of pyridoxal phosphate. The Km for this cofactor was 2.7 x 10-7 m. Inorganic phosphate was found to be a competitive inhibitor with an apparent Ki of 1.3 x 10-3 m. None of the cations or anions tested was found to be essential for enzyme activity or to have an ability to activate the enzyme. The enzyme was inactivated by heavy metal cations, sulfate ions, and cyanide ions. Inhibition of p-chloromercuribenzoate could be reversed by dithiothreitol. Substrate specificity was tested with a variety of compounds; ethanolamine, ethylphosphate, ethylamine, ethylenimine, and 2-aminoethylphosphonate were not substrates for the enzyme. The approximate molecular weight as estimated by Sephadex G-200 chromatography was 168,000. An Arrhenius plot of enzyme activity showed a discontinuity with the energy of activation being 18,700 calories below 14° and 10,700 calories at higher temperature. By means of O-phosphorylethanolamine labeled with 14C in the amino carbon, it was shown by oxidation and Schmidt degradation of the enzymatically formed acetaldehyde that the 14C was solely in the carbonyl carbon of the product. These data serve to confirm the mechanism of this reaction as an initial β-elimination of Pi of the O-phosphorylethanolamine-pyridoxal phosphate complex. The purpose of this paper is to report the partial purification of the enzyme from liver which catalyzes the reaction H3N+CH2CH2OPO3- (pyridoxal-P)/→ NH4+ + CH2CHO + Pi and to describe some of its physical and chemical properties. In addition, studies utilizing 2-14C-O-phosphorylethanolamine indicate that the mechanism of the reaction catalyzed by the enzyme is that of a phospho-lyase (Sprinson, D. B., and Weliky, I., Biochem. Biophys. Res. Commun., 36, 866 (1969)) rather than an ammonia lyase as was originally indicated (Fleshood, H. L., and Pitot, H. C., Biochem. Biophys. Res. Commun., 36, 110 (1969)).

The purification and properties of oxidized derivatives of l-histidine ammonia-lyase

1. 1. l-Histidine ammonia-lyase ( l-histidine ammonia-lyase, EC 4.3.1.3) purified from Pseudomonas fluorescens is unstable in air. Treatment of the enzyme with 5,5′-dithio-bis-(2-nitrobenzoic acid) produces a stable derivative which retains a fraction of the original activity and which may be restored to full activity by treatment with dithiothreitol. The oxidation of histidine ammonia-lyase by O 2 and by H 2O 2 has also been investigated. 2. 2. The kinetic behavior of 5,5′-dithio-bis-(2-nitrobenzoic acid)-oxidized histidine ammonia-lyase (TNB-histidine ammonia lyase) and its response to photosensitized oxidation and to thermal inactivation were all consistent with the existence of two classes of active sites. 3. 3. EDTA was a competitive inhibitor of reduced histidine ammonia-lyase but had no effect on TNB-histidine ammonia-lyase. The maximum inhibition which could be achieved with EDTA was close to 90%. The interaction of EDTA and the reduced enzyme was rapid in the absence of l-histidine but was perceptably slow in its presence. 4. 4. The time-course of the inhibition of histidine ammonia-lyase by borohydride was investigated under conditions which achieved a steady-state level of borohydride in the reaction mixture. The effects of pH and of a variety of small molecules on the borohydride-induced inactivation of the enzyme were explored. These data appear to be consistent with the proposal of an azomethine function at the active site.

Biochemical basis of resistance in eggplant (<i>Solanum melongena</i> L.) to <i>Leucinodes orbonalis</i> Guenee and their correlation with shoot and fruit infestation

Studies on the biochemical basis of resistance to Leucinodes orbonalis Guenee and their correlation with shoot and fruit borer damage in five selected brinjal genotypes done at Tamil Nadu Agricultural University, India during June to December 2005 showed that both shoot and fruit of less susceptible genotypes had the higher amount of poly phenol oxidase (PPO), phenylalanine ammonium lyase (PAL) and lignin and lower amount of reducing sugar. Significant negative correlation was found between percent infestation (shoot and fruit) with PPO, PAL and lignin content, whereas it was positively correlated with reducing sugar content. Among the biochemical constituents, PPO, PAL and lignin contents were negatively correlated with reducing sugar but PPO were positively correlated with PAL and lignin content and vice-versa. Keywords: Biochemical basis; resistacace; eggplant; Leucinodes orbonalis. DOI: 10.3329/bjar.v35i1.5876Bangladesh J. Agril. Res. 35(1) : 149-155, March 2010

Transaminierung und desaminierung von phenylalanin in wurzeln von Zea mays

l-phenylalanine transaminase and ammonia lyase were isolated from cell-free extracts of 3-day-old Zea mays roots and fractionated with (NH 4) 2SO 4 and Sephadex G 200. The pH optima and temperature stability of both enzymes and the substrate specificity of the transaminase were determined.