Kinetic analysis of the inhibition of phenylalanine ammonia-lyase by 2-aminoindan-2-phosphonic acid and other phenylalanine analogues

The conformationally restricted phenylalanine analogue 2-aminoindan-2-phosphonic acid (AIP) inhibits phenylalanine ammonia-lyase (PAL) competitively in a time-dependent manner. This phenomenon was investigated in more detail with the heterologously expressed, highly purified homotetrameric PAL-1 isozyme from parsley. The kinetic analysis revealed that the enzyme-inhibitor complex is formed in a single "slow" step with an association rate of k(2)=2.6+/-0.04 10(4) M(-1) s(-1). The inhibition is reversible with a dissociation rate of k(-2)=1.8+/-0.04 10(-4) s(-1) and an equilibrium constant of K(i)=7+/-2 nM. The previously described PAL inhibitor (S)-2-aminooxy-3-phenylpropanoic acid [(S)-AOPP] was also found to be a slow-binding inhibitor of PAL-1. The carboxyl analogue of AIP, 2-aminoindan-2-carboxylic acid, served as a substrate of PAL-1 and was converted to indene-2-carboxylic acid.     

Inhibition of phenylalanine ammonia-lyase activity causes the depression of lignin accumulation of secondary wall thickening in isolatedZinnia mesophyll cells

Summary Isolated mesophyll cells ofZinnia elegans L. cv. Canary Bird differentiate into tracheary elements in differentiation (D) medium. These elements develop lignified secondary wall thickenings. The influence of 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase (PAL), on lignification ofZinnia tracheary elements was examined. The mesophyll cells were cultured in D and AIP media. The latter medium, in which 100 M AIP was added to the D medium, inhibited PAL activity, though the differentiation proceeded. Morphological differences of secondary wall thickenings cultured in these two types of media were investigated under an UV microscope and a transmission electron microscope. The secondary wall thickenings at 96 h in the D medium showed strong UV absorption. The fibrillar structure of the thickenings observed clearly at 72 h was covered with electron opaque materials by 96 h. The secondary wall thickenings at 96 h in the AIP medium showed weak UV absorption. The thickenings at 96 h had a cracked appearance. Furthermore, the thickenings showed a little irregular or wavy arrangement of cellulose microfibrils and had many pores and spaces between microfibrils. From these results, the role of lignin accumulation in the formation of secondary wall thickenings was discussed.Abbreviations AIP 2-aminoindan-2-phosphonic acid - PAL phenylalanine ammonia-lyase     

Inhibitors of phenylalanine ammonia-lyase (PAL): synthesis and biological evaluation of 5-substituted 2-aminoindane-2-phosphonic acids

A series of 5-substituted derivatives of the potent phenylalanine ammonia-lyase (PAL) inhibitor 2-aminoindane-2-phosphonic acid (AIP; 2) were synthesized. The AIP analogues 3-7, with additional NO2, NH2, Me, Br, and OH groups, respectively, were tested as in vitro inhibitors of buckwheat PAL, and as in vivo inhibitors of anthocyanin biosynthesis. Within this series, the racemic 5-bromo (6) and 5-methyl (7) congeners were biologically most active (Table), although being ca. one order of magnitude less potent than AIP proper.     

Effect of yeast elicitor on the secondary metabolism of Ti-transformed Salvia miltiorrhiza cell suspension cultures

 Salvia miltiorrhiza contains two groups of biologically active secondary metabolites termed phenolic compounds (e.g. rosmarinic acid) and tanshinones (e.g. cryptotanshinone). Their roles in plant defense responses were examined using a simplified system consisting of a yeast elicitor and a Ti C58 transformed S. miltiorrhiza cell line. Both dosage and time course studies were carried out on the effects of yeast elicitor on the formation of rosmarinic acid and cryptotanshinone. It was found that the yeast elicitor reduced the constituent level of rosmarinic acid (from ca. 5% to ca. 3.0% of dry cell weight) whereas the level of cryptotanshinone was enhanced greatly (from a negligible amount to ca. 20 mg/l). These results suggest that in S. miltiorrhiza, rosmarinic acid and cryptotanshinone may take part in plant passive and active defense responses, respectively, against pathogen attack. Cryptotanshinone was identified as a phytoalexin in S. miltiorrhiza for the first time. Results of the treatment of cell cultures with 2-aminoindan-2-phosphonic acid, a highly specific and potent inhibitor of phenylalanine ammonia-lyase (EC 4.3.1.5), indicated that this compound did not inhibit yeast elicitor induced tanshinone formation, but did inhibit rosmarinic acid biosynthesis. Received: 22 May 1999 / Revision received: 27 August / Accepted: 1 September 1999     

Photocontrol of chlorogenic acid biosynthesis in potato tuber discs

The appearance of phenylalanine ammonia-lyase activity and the accumulation of chlorogenic acid in potato tuber discs are stimulated by illumination with white light, whereas the appearance of cinnamic acid 4-hydroxylase activity is unaffected by illumination. The photosensitive step in chlorogenic acid biosynthesis may be by-passed by treatment of discs with exogenous supplies of cinnamic acid, whereas treatment of discs with phenylalanine does not isolate the photosensitive step. Therefore, the site of photocontrol of chlorogenic acid biosynthesis in potato tuber discs is the reaction catalysed by phenylalanine ammonia-lyase. Cinnamic acid 4-hydroxylase activity in vitro is unaffected by p-coumaric acid, caffeic acid or chlorogenic acid. Phenylalanine ammonia-lyase activity in vitro is sensitive to inhibition by cinnamic acid. The in vitro properties of the two enzymes are also consistent with the hypothesis that phenylalanine ammonia-lyase rather than cinnamic acid 4-hydroxylase is important in the regulation of chlorogenic acid biosynthesis in potato tuber discs.     

Effects of competitive inhibitors of phenylalanine ammonia-lyase on the levels of phenylpropanoid enzymes in Solatium tuberosum

Abstract The accumulation of chlorogenic acid in illuminated discs of Solanum tuberosum tuber tissue is accompanied by rapid but transient increases in the activity levels of the biosynthetic enzymes phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase and hydroxycinnamoyl-CoA : quinate hydroxycinna-moyl transferase. Exogenous D-phenylalanine and L-α-aminooxy-β-phenylpropionic acid, competitive inhibitors of phenylalanine ammonia-lyase, inhibit the accumulation of chlorogenic acid and presumably reduce the endogenous pools of pathway intermediates such as cinnamic acid. These treatments prolong the phase of increase in phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase activities and indicate that product feedback modulation is important in maintaining the interrelationship between the levels of these two enzymes during the later stages of induction. In contrast,L-α-aminooxy-β-phenylpropionic acid inhibits the development of hydroxycinnamoyl transferase in illuminated discs supporting the idea that the light-stimulated increase in phenylalanine ammonia-lyase activity causes an increase in cinnamic acid production which mediates the light-stimulated increase in hydroxycinnamoyl transferase activity.     

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 Mg²⁺ 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/cm³), and also in fractions at higher densities (d=1.12–1.13 and 1.15 g/cm³).Abbreviations PAL L-phenylalanine ammonia-lyase - Tris tris-(hydroxymethyl)aminomethane - EDTA ethylenediamine tetraacetic acid - ATPase ATP phosphohydrolase     

Novel photoreactive cinnamic acid analogues to elucidate phenylalanine ammonia-lyase

4-[3-(Trifluoromethyl) diazirinyl] cinnamic acid derivatives were synthesized to elucidate properties of phenylalanine ammonia-lyase (PAL). 2-Methoxy and 2-biotinylated alkoxy compounds have inhibitory activity on the formation of phenylalanine from cinnamic acid. Specific photolabeling of the enzyme was detected using biotinylated derivatives without the use of radioisotopes. The results indicated that the 4-[3-(trifluoromethyl) diazirinyl] skeleton will be a suitable photoreactive compound to elucidate regulation of phenylpropanoid biosynthesis.     

Localization of N-Methyl-d-Aspartate Receptors in the Rat Striatum: Effects of Specific Lesions on the [3H]3-(2-Carboxypiperazin-4-yl)propyl-1-Phosphonic Acid Binding

The binding of [3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ([3H]CPP), a rigid analogue of 2-amino-7-phosphonoheptanoic acid (AP7) and reported to be a selective N-methyl-D-aspartate (NMDA) antagonist, was studied in rat striatal membranes using a centrifugation procedure to separate bound and free radioligand. [3H]CPP bound with high affinity (KD = 272 nM) in a saturable, reversible, and protein concentration-dependent manner. Specific binding was suggested to involve a single class of noninteracting binding sites. The most potent [3H]CPP binding inhibitors tested were CPP, L-glutamate, 2-amino-5-phosphonovalerate, and AP7. NMDA, L-aspartate, and alpha-aminoadipate were also shown to be efficient in inhibiting the binding, whereas quisqualate, D,L-2-amino-4-phosphonobutyrate, kainate, L-glutamate diethylester, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid were found to be essentially inactive. These data are therefore consistent with the view that [3H]CPP selectively binds to NMDA receptors in the rat striatum. Lesions of intrastriatal neurons using local injections of kainic acid revealed a marked decrease in [3H]CPP binding, suggesting an almost exclusively postsynaptic location of binding sites in the striatum. Conversely, bilateral lesion of corticostriatal glutamatergic fibers resulted in an increased number of [3H]CPP striatal binding sites, providing evidence for a putative supersensitivity response to this striatal deafferentation. Interestingly, lesion of the nigrostriatal dopaminergic neurons using intranigral 6-hydroxydopamine injections resulted, 2-3 weeks later, in a similar increase in the number of [3H]CPP striatal binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Salicylates of intact Salix myrsinifolia plantlets do not undergo rapid metabolic turnover

Salicylates, the main phenolic glucosides of northern willow (Salix spp.), play an important role in plant-herbivore interactions. Salicylates are labile metabolites that are thought to undergo metabolic turnover. Salicylates are synthesized from phenylalanine (Phe) via the shikimate pathway. 2-Aminoindan-2-phosphonic acid (AIP), a strong inhibitor of Phe ammonia-lyase (EC 4.3.1.5), was used to block the biosynthesis of salicylates. The aim of this study was to investigate long-term turnover of salicylates in intact micropropagated plantlets of Salix myrsinifolia Salisb. The biosynthesis of salicylates was inhibited efficiently but not completely by 30 microM 2-aminoindan-2-phosphonic acid. Inhibitor treatment, aside from leading to a high accumulation of Phe, also led to an increase in tyrosine and tryptophan, indicating that 2-aminoindan-2-phosphonic acid may also inhibit enzymes other than Phe ammonia-lyase. Salicylates were shown to be unexpectedly stable metabolites that did not undergo marked metabolic turnover in intact plants; in leaves no significant turnover occurred, and in the stems the five salicylates studied were turned over slowly, with half-lives of 11 to 25 d. The total amount of salicylate in mature shoots decreased only 0.6% per day.     

Hydroxylated Analogues of 5-Aminovaleric Acid as 4-Aminobutyric AcidB Receptor Antagonists: Stereostructure-Activity Relationships

The (R) and (S) forms of 5-amino-2-hydroxyvaleric acid (2-OH-DAVA) and 5-amino-4-hydroxyvaleric acid (4-OH-DAVA) were designed as structural hybrids of the 4-aminobutyric acidB (GABAB) agonist (R)-(-)-4-amino-3-hydroxybutyric acid [(R)-(-)-3-OH-GABA] and the GABAB antagonist 5-aminovaleric acid (DAVA). (S)-(-)-2-OH-DAVA and (R)-(-)-4-OH-DAVA showed a moderately potent affinity for GABAB receptor sites in rat brain and showed GABAB antagonist effects in a guinea pig ileum preparation. The respective enantiomers, (R)-(+)-2-OH-DAVA and (S)-(+)-4-OH-DAVA, were markedly weaker in both test systems. All four compounds were weak inhibitors of GABAA receptor binding in rat brain, and none of them significantly affected synaptosomal GABA uptake. Based on molecular modeling studies it has been demonstrated that low-energy conformations of (R)-(-)-3-OH-GABA, (S)-(-)-2-OH-DAVA, and (R)-(-)-4-OH-DAVA can be superimposed. These conformations may reflect the shapes adopted by these conformationally flexible compounds during their interaction with GABAB receptors. The present studies emphasize the similar, but distinct, constraints imposed on agonists and antagonists for GABAB receptors.     

Control of phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase in gherkin tissues

Blue light mediates a transient increase in the extractable activity of phenylalanine ammonia-lyase from both cotyledons and hypocotyls of etiolated gherkin seedlings, but concurrent changes in extractable cinnamic acid 4-hydroxylase activity only occur in cotyledons. Excision, followed by incubation in the dark, also results in stimulation of the lyase activity in both tissues, but the hydroxylase activity is only stimulated in cotyledons, again concurrently with the lyase. Stimulated levels of hydroxycinnamic acid esters are, however, only formed following light treatment, indicating the presence of another light-sensitive step in their biosynthesis. Treatment of gherkin tissues with 2-aminooxyacetic acid or α-aminooxy-β-phenylpropionic acid inhibits phenylalanine ammonia-lyase activity in situ, reduces the accumulation of hydroxycinnamic acid esters and presumably reduces the endogenous cinnamic acid pool. This treatment increases extractable lyase activity and delays its peak in activity. In cotyledons, these changes in the lyase are associated with concurrent and similar changes in extractable hydroxylase activity, whilst in hypocotyls the hydroxylase is relatively unaffacted. The increase in phenylalanine ammonia-lyase activity following excision of cotyledons and hypocotyls is prevented by cinnamic acid; in cotyledons the hydroxylase is similarly affected, but after a longer lag. Thus whilst cinnamic acid can modify the extractable activity of the lyase, it cannot itself mediate changes in the extractable activity of the hydroxylase.     

Induction of rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures by yeast extract

Summary A transient increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after addition of yeast extract (YE) to the suspension cultures, reaching a maximum at 24 hr. The highest increase of the RA content (2.5-fold) was obtained when 6-day-old cells in the exponential growth phase were treated with YE. Preceding the induced RA accumulation, phenylalanine ammonia-lyase (PAL) activity increased rapidly, whereas tyrosine aminotransferase (TAT) activity was largely unaffected by the treatment. The incorporation of both ¹⁴C-phenylalanine and ¹⁴C-tyrosine into RA was enhanced in the YE-treated cells, consistent with increased synthesis of the ester.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - PAL phenylalanine ammonia-lyase - TAT tyrosine aminotransferase - RA rosmarinic acid - YE yeast extract     

Inhibition of phenylalanine ammonia-lyase by cinnamic acid derivatives and related compounds

Thirty-five derivatives of cinnamic acid and related compounds were tested for inhibition against phenylalanine ammonia-lyase (PAL) derived from sweet potato, pea and yeast. Caffeic and gallic acids showed inhibition against PAL originating from higher plants, but not against yeast PAL. In contrast, yeast PAL was specifically inhibited by p-hydroxycinnamic and p-hydroxybenzoic acids. The results suggest that caffeic and gallic acids may act as regulatory substances in phenylpropanoid metabolism in higher plants. Inhibition experiments with synthetic cinnamic acid derivatives have revealed that the presence of a hydrophobic aromatic ring, α,β-double bond and carboxyl group is essential for inhibitory activity. 2-Naphthoic acid which fulfills these structural requirements showed a strong inhibition. The size and shape of the active site is discussed from structure-activity relationships of cinnamic acid derivatives. o-Chlorocinnamic acid, one of the strongest inhibitors found in this study showed an inhibitory effect on the growth of the roots of rice seedlings.     

Genetic engineering of shikonin biosynthesis hairy root cultures of Lithospermum erythrorhizon transformed with the bacterial ubiC gene

The biosynthetic pathway to 4-hydroxybenzoate (4HB), a precursor of the naphthoquinone pigment shikonin, was modified in Lithospermum erythrorhizon hairy root cultures by introduction of the bacterial gene ubiC. This gene of Escherichia coli encodes chorismate pyruvate-lyase (CPL), an enzyme that converts chorismate into 4HB and is not normally present in plants. The ubiC gene was fused to the sequence for a chloroplast transit peptide and placed under control of a constitutive plant promoter. This construct was introduced into L. erythrorhizon by Agrobacterium rhizogenes-mediated transformation.The resulting hairy root cultures showed high CPL activity. 4HB produced by the CPL reaction was utilized for shikonin biosynthesis, as shown by in vivo inhibition of the native pathway to 4HB with 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase. A feeding experiment with [1,7-13C2]shikimate showed that in the absence of AIP the artificially introduced CPL reaction contributed ca. 20% of the overall 4HB biosynthesis in the transgenic cultures. ubiC transformation did not lead to a statistically significant increase of shikonin formation, but to a 5-fold increase of the accumulation of menisdaurin, a nitrile glucoside which is presumably related to aromatic amino acid metabolism.     

Relationship between the enzymatic browning and phenylalanine ammonia-lyase activity of cut lettuce, and the prevention of browning by inhibitors of polyphenol biosynthesis.

Cut lettuce stored at 4 degrees C gradually turned brown on the cut section after several days of storage. Three factors for enzymatic browning, the polyphenol content, polyphenol oxidase activity, and phenylalanine ammonia-lyase (PAL) activity, were examined during the cold storage of cut lettuce. A relationship between the browning and PAL activity was apparent. We tried to prevent this browning by using the two enzyme inhibitors, 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of the phenylpropanoid pathway, and glyphosate, an inhibitor of the shikimate pathway. AIP and glyphosate significantly inhibited the browning of cut lettuce. The polyphenol content and PAL activity were both reduced by the treatment with AIP. These results show that regulating the biosynthesis of polyphenols is essential to prevent the browning of cut lettuce.     

Synthesis and biological evaluation of 4-fluoroproline and 4-fluoropyrrolidine-2-acetic acid derivatives as new GABA uptake inhibitors

Preparation for the N-alkylated derivatives of enantiomerically pure (2S)-4-fluoroproline and (2S)-4-fluoropyrrolidine-2-acetic acid is described. The final compounds were evaluated as potential GAT-1 uptake inhibitors via cultured cell lines expressing mouse GAT-1. Compared with their corresponding 4-hydroxy compounds, these derivatives exhibited slight improvement on their inhibitory potency, but still much weaker than their corresponding compounds with no substituents at the C-4 of the pyrrolidine moiety, with the most potent affinity being about 1/15 fold as that of Tiagabine. The drastic decrease of their affinity may arise from sharp reduction of their basicity due to strong inductive effect of the 4-fluorine. However the configuration of the C-4 linking fluorine did not have much influence on their affinity for GAT-1.     

3-[(±)-2-Carboxypiperazin-4-yl]propyl-1-Phosphonic Acid Recognizes Two N-Methyl-D-Aspartate Binding Sites in Rat Cerebral Cortex Membranes

Binding of 3-[(+-)-2-carboxypiperazin-4-yl][3H]-propyl-1-phosphonic acid ([3H]CPP), a competitive inhibitor of N-methyl-D-aspartate (NMDA), has been studied in synaptic plasma membranes from rat cerebral cortex. Computer analysis of saturation and homologous displacement isotherms deriving from these plasma membranes indicated the existence of two binding sites: a specific, saturable, high-affinity binding site with a pKD value of 7.53 +/- 0.03 (29.5 nM) and a maximum binding value (Bmax) of 2.25 +/- 0.36 pmol/mg of protein, and a low-affinity site with a KD of approximately 600 nM and a Bmax of 7.0 pmol/mg of protein. It is argued that, in the light of current literature evidence, the low-affinity binding site may represent an agonist-dependent receptor, linked to physiological processes such as neurotransmitter release and channel regulation, whereas the high-affinity binding site may be linked to an antagonist-preferred receptor, for which no function has yet been reported.     

Superinduction of phenylalanine ammonia-lyase in gherkin hypocotyls caused by the inhibitor, L-alpha-aminooxy-beta-phenylpropionic acid.

The extractable activity of L-phenylalanine ammonia-lyase (EC 4.3.1.5) and the concentration of sugar esters of p-coumaric and ferulic acids in the hypocotyls of etiolated gherkin seedlings increase upon irradiation with white light. Treatment of intact seedlings with the phenylalanine ammonia-lyase inhibitors alpha-aminooxyacetic acid and L-alpha-aminooxy-beta-phenylpropionic acid during illumination causes enhanced formation of the lyase and reduces the accumulation of hydroxycinnamic acids. Enzyme activity in excised hypocotyl segments floating on buffer increases in the dark as well as in the light, while hydroxycinnamic acids accumulate only in the light. Phenylalanine ammonia-lyase formation in the segments is inhibited by cinnamic acid and, to a lesser extent, p-coumaric acid, while it is slightly enhanced by caffeic acid and is not affected by ferulic acid. Aminooxyphenylpropionate dramatically promotes phenylalanine ammonia-lyase formation in the segments in darkness and light prevents the accumulation of hydroxycinnamic acids in the light. Aminooxyphenylpropionate does not, however, affect the time course of apparent lyase formation and decay. Cinnamic acid, the product of the lyase reaction, antagonizes the effect of aminooxyphenylpropionate. It is proposed that the reaction product(s) are involved to some extent in the regulation of the pool of active lyase in the hypocotyl tissue.     

Rosmarinic acid production in Coleus cell cultures

Cell suspension cultures of Coleus blumei Benth. have been found to accumulate 8–11% of their dry weight as rosmarinic acid (-O-caffeoyl-3,4-dihydroxyphenyl-lactic acid). Actively-growing tissue converts >20% of exogenously supplied phenylalanine and tyrosine to the caffeoyl ester and this high rate of synthesis coincides with an increase in phenylalanine ammonia-lyase specific activity. Administration to the cultures of known phenylpropanoid precursors of rosmarinic acid failed to enhance the latter's production and in some cases inhibited it.Abbreviations RA rosmarinic acid (-O-caffeoyl-3,4-dihydroxyphenyllactic acid - DOPA dihydroxyphenylalanine - PAL phenylalanine ammonialyase - DOPL dihydroxyphenyl-lactic acid