Article

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

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

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.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... Bryant et al. 1983; Loomis 1932, 1953; Herms and Mattson 1992) in plants. Different inhibitors of PAL have been tested (see Appert et al. (2003) for an overview), and among these 2-aminoindan-2-phosphonic acid monohydrate (AIP) was found to be particularly effective and selective, both in vivo and in vitro (Zonánd Amrhein 1992; Zonét al. 2004). AIP is a slow binding inhibitor of PAL, and the inhibition has been shown to be reversible in vitro in a pure, clearly defined PAL preparation (Appert et al. 2003). ...
... Different inhibitors of PAL have been tested (see Appert et al. (2003) for an overview), and among these 2-aminoindan-2-phosphonic acid monohydrate (AIP) was found to be particularly effective and selective, both in vivo and in vitro (Zonánd Amrhein 1992; Zonét al. 2004). AIP is a slow binding inhibitor of PAL, and the inhibition has been shown to be reversible in vitro in a pure, clearly defined PAL preparation (Appert et al. 2003). AIP has been shown to inhibit the accumulation of many kinds of phenolic compounds (Zonánd Amrhein 1992; Reuber et al. 1993; Gitz et al. 1998; Ruuhola and Julkunen-Tiitto 2000, 2003). ...
Article
Full-text available
Phenylalanine ammonia lyase (PAL) plays a key role in phenylpropanoid metabolism, catalyzing the deamination of phenylalanine (Phe) to form trans-cinnamic acid. Inhibitors of PAL have been used to study the physiological role of the different compounds derived from trans-cinnamic acid, and to test theories about a trade-off between growth and defence in plants. In a previous study with birch (Betula pubescens Ehrh.) seedlings, the PAL inhibitor 2-aminoindane-2-phosphonic acid monohydrate (AIP) caused an accumulation of Phe and a strong decrease in the quantity of simple phenolics, soluble condensed tannins and growth, whereas flavonol glycosides were generally not affected. The present study demonstrates restoration of secondary metabolism in the previously AIP treated birch seedlings. Our results indicate that Phe accumulated during PAL inhibition could be partly used to increase the content of the phenolic acids, flavan-3-ols and to some extent the soluble condensed tannins. Seedling growth also increased when the supply of PAL inhibitor ceased. We thereby show that the inhibition of PAL by AIP in vivo is reversible, at least for moderate AIP concentrations and the rate of restoration is dependent on the inhibitor concentration.
... While cultural factors such as light exposure, temperature, plant nutrition, and ontological development are known to influence this pathway, numerous factors were evaluated in the current study and were insufficient to facilitate a reproducible protoplast isolation protocol from American elm (data not shown). A number of competitive PAL inhibitors, namely 2-aminoindane-2-phosphonic acid (AIP [30,31]), (S)-2-aminooxy-3-phenylpropionic acid (AOPP, notation (S) and L are equal [32]) and O-benzylhydroxylamine (OBHA [33]) have been shown to significantly reduce the production of phenylpropanoids in a variety of species. In a previous study with Lycopersicon esculentum suspension cultures, the addition of AIP to the medium effectively reduced the cells ability to accumulate wall bound phenolics when challenged with a fungal elicitor [34]. ...
... Addition of AOPP resulted in a modest increase in protoplast isolation while OBHA had no beneficial effect (data not shown). These data support previous studies that found AIP to be more effective at inhibiting PAL than the other two compounds [31]. ...
Article
Full-text available
Protoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L.) was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall re-synthesis and cell division. This study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L.) leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 μM), an inhibitor of phenylalanine ammonia lyase (PAL), reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (±3.27) in controls to 65.3% (±4.60). Protoplasts isolated from callus grown in 100 μM AIP developed cell walls by day 2, had a division rate of 28.5% (±3.59) by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose. This study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated protoplasts survived electrofusion and viable heterokaryons were produced. Together, these results provide the first evidence of sustained cell division, callus regeneration, and potential application of somatic cell fusion in American elm, suggesting that this source of protoplasts may be ideal for genetic manipulation of this species. The technological advance made with American elm in this study has potential implications in other woody species for fundamental and applied research which require availability of viable protoplasts.
... Notably, HAL and PAL samples bearing modified cofactors are catalytically inactive [18,19]. In crystal soaking experiments of RsTAL with the PAL product cinnamic acid or the PAL-specific inhibitor, 2-aminoindan-2phosphonic acid (AIP) [23], no significant binding was observed, in accord with the poor turnover of L-Phe by RsTAL and the weak inhibition of RsTAL activity by AIP (see discussion below and Table 2). ...
... Indeed, the catalytic efficiency of the PAL activity for the H89F mutant (k cat /K m 0.019 s −1 μM −1 ) is only slightly lower than that of TAL activity for wild-type RsTAL (0.058 s −1 μM −1 ), and it exceeds the catalytic efficiency of some native PALs (Table 2). In addition, the differing kinetic specificities of the wild-type and mutant RsTALs are further substantiated by the relative susceptibilities to inhibition by AIP, a PALspecific inhibitor [23]. For wild-type RsTAL, activity with L-Tyr is unaffected by AIP, whereas activity with LPhe is inhibited (K i =16.3 μM). ...
Article
Aromatic amino acid ammonia-lyases catalyze the deamination of L-His, L-Phe, and L-Tyr, yielding ammonia plus aryl acids bearing an alpha,beta-unsaturated propenoic acid. We report crystallographic analyses of unliganded Rhodobacter sphaeroides tyrosine ammonia-lyase (RsTAL) and RsTAL bound to p-coumarate and caffeate. His 89 of RsTAL forms a hydrogen bond with the p-hydroxyl moieties of coumarate and caffeate. His 89 is conserved in TALs but replaced in phenylalanine ammonia-lyases (PALs) and histidine ammonia-lyases (HALs). Substitution of His 89 by Phe, a characteristic residue of PALs, yields a mutant with a switch in kinetic preference from L-Tyr to L-Phe. Structures of the H89F mutant in complex with the PAL product, cinnamate, or the PAL-specific inhibitor, 2-aminoindan-2-phosphonate (AIP), support the role of position 89 as a specificity determinant in the family of aromatic amino acid ammonia-lyases and aminomutases responsible for beta-amino acid biosynthesis.
... PAL catalyzes the conversion of phenylalanine to t-cinnamic acid from which salicylic acid (SA) is derived. Aminooxyacetic acid (AOA) and L-2-aminooxy-3-phenylpropionic acid (AOPP), PAL inhibitors (Kessmann et al., 1990;Appert et al., 2003), inhibit stress-induced flowering in L. paucicostata and pharbitis, and the inhibitory effect of AOA is negated by SA (Wada et al., 2010b;Shimakawa et al., 2012). The endogenous SA content increases in the plants flowered by stress (Shimakawa et al., 2012;Wada et al., 2014). ...
... More important unknown factors could be involved in P. frutescens flowering. et al., 1990;Appert et al., 2003). The inhibition of flowering by AOPP and AOA in P. frutescens may be through mechanisms that are different from PAL inhibition. ...
... As shown in Fig. 7, among the 9 compounds tested, there are four types of inhibition: competitive (coniferyl alcohol, coniferyl aldehyde, salidroside), uncompetitive (coumaric acid, cinnamyl alcohol, chlorogenic acid), mixed (cinnamic acid) and no inhibition (tyrosol and caffeic acid). The K i of competitive inhibition compounds was calculated as K i calculation of 2-aminoindan-2-phosphonic acid (AIP) that is the competitive PAL specific inhibitor [19,20], and in case of uncompetitive inhibition compounds, the K i was calculated using the formula V max 0 = V max /(1 ? [i]/K i ). ...
Article
Full-text available
We cloned the gene, CdPAL1, from Cistanche deserticola callus using RACE PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes from other plant species. The gene shows high homology to other known PAL genes registered in GenBank. The recombinant protein exhibited Michaelis-Menten kinetics with a Km of 0.1013 mM, Vmax of 4.858 μmol min(-1), Kcat of 3.36 S(-1), and Kcat/Km is 33,168 M(-1) S(-1). The enzyme had an optimal pH of 8.5 and an activation energy of 38.92 kJ mol(-1) when L-Phenylalanine was used as a substrate; L-tyrosine cannot be used as substrate for this protein. The optimal temperature was 55°C, and the thermal stability results showed that, after a treatment at 70°C for 20 min, the protein retained 87% activity, while a treatment at 75°C for 20 min resulted in a loss of over 85% of the enzyme activity. Treatment with heavy metal ions (Hg2+, Pb2+, and Zn2+) showed remarkable inhibitory effects. Among the intermediates from the lignin (cinnamyl alcohol, cinnamyl aldehyde, coniferyl aldehyde, coniferyl alcohol), phenylpropanoid (cinnamic acid, coumaric acid, caffeic acid, and chlorogenic acid) and phenylethanoid (tyrosol and salidroside) biosynthetic pathways, only cinnamic acid showed strong inhibitory effects against CdPAL1 activity with a Ki of 8 μM. Competitive inhibitor AIP exhibited potent inhibition with Ki=0.056 μM.
... The effect of the addition of the PAL inhibitor AIP is more difficult to interpret as it clearly reduces but does not eliminate the induction of PR1 after H. parviporum infection. AIP acts as reversible competitive inhibitor; it is possible that a certain PAL activity that could convert phenylalanine to cinnamic acid remains after the treatment with AIP (Appert et al. 2003), and that this residual PAL activity produces sufficient cinnamic acid for some pathogen-induced SA production. Most likely, however, the JA-mediated induction of PR1 in Norway spruce is not dependent on PAL-mediated production of cinnamic acid. ...
Article
Full-text available
A key tree species for the forest industry in Europe is Norway spruce [Picea abies (L.) Karst.]. One of its major diseases is stem and butt rot caused by Heterobasidion parviporum (Fr.) Niemelä & Korhonen, which causes extensive revenue losses every year. In this study, we investigated the parallel induction of Norway spruce genes presumably associated with salicylic acid- and jasmonic acid/ethylene-mediated signalling pathways previously observed in response to H. parviporum. Relative gene expression levels in bark samples of genes involved in the salicylic acid- and jasmonic acid/ethylene-mediated signalling pathways after wounding and inoculation with either the saprotrophic biocontrol fungus Phlebiopsis gigantea or with H. parviporum were analysed with quantitative PCR at the site of the wound and at two distal locations from the wound/inoculation site to evaluate their roles in the induced defence response to H. parviporum in Norway spruce. Treatment of Norway spruce seedlings with methylsalicylate, methyljasmonate and inhibitors of the jasmonic acid/ethylene signalling pathway, as well as the Phenylalanine ammonia lyase inhibitor 2-aminoindan-2-phosphonic acid were conducted to determine the responsiveness of genes characteristic of the different pathways to different hormonal stimuli. The data suggest that jasmonic acid-mediated signalling plays a central role in the induction of the genes analysed in this study irrespective of their responsiveness to salicylic acid. This may suggest that jasmonic acid-mediated signalling is the prioritized module in the Norway spruce defence signalling network against H. parviporum and that there seems to be no immediate antagonism between the modules in this interaction.
... The phenylalanine structural analog, 2-aminoindane-2phosphonic acid (AIP), is a well documented competitive inhibitor of PAL both in vitro and in vivo, and provides an effective means to prevent the synthesis of phenolic compounds in a variety of systems [21][22][23][24][25][26][27][28][29][30][31][32][33]. Inhibition of PAL through the use of AIP has the potential to significantly reduce the biosynthesis of phenolic compounds, and could represent a novel approach to controlling oxidative browning in plant tissue culture. ...
Article
Full-text available
Oxidative browning is a common and often severe problem in plant tissue culture systems caused by the accumulation and oxidation of phenolic compounds. The current study was conducted to investigate a novel preventative approach to address this problem by inhibiting the activity of the phenylalanine ammonia lyase enzyme (PAL), thereby reducing the biosynthesis of phenolic compounds. This was accomplished by incorporating 2-aminoindane-2-phosphonic acid (AIP), a competitive PAL inhibitor, into culture media of Artemisia annua as a model system. Addition of AIP into culture media resulted in significant reductions in visual tissue browning, a reduction in total phenol content, as well as absorbance and autoflourescence of tissue extracts. Reduced tissue browning was accompanied with a significant increase in growth on cytokinin based medium. Microscopic observations demonstrated that phenolic compounds accumulated in discrete cells and that these cells were more prevalent in brown tissue. These cells were highly plasmolyzed and often ruptured during examination, demonstrating a mechanism in which phenolics are released into media in this system. These data indicate that inhibiting phenylpropanoid biosynthesis with AIP is an effective approach to reduce tissue browning in A. annua. Additional experiments with Ulmus americana and Acer saccharum indicate this approach is effective in many species and it could have a wide application in systems where oxidative browning restricts the development of biotechnologies.
... Furthermore, PAL catalyzes the conversion of phenylalanine to tcinnamic acid, and salicylic acid (SA) is derived from t-cinnamic acid (Yalpani et al., 1993). We found that aminooxyacetic acid (AOA) and l-2-aminooxy-3-phenylpropionic acid, which function as PAL inhibitors (Kessmann et al., 1990;Appert et al., 2003), inhibited stress-induced flowering. This inhibitory effect was negated by tcinnamic acid and SA in pharbitis (Hatayama and Takeno, 2003;Wada et al., 2010b). ...
Article
The involvement of salicylic acid (SA) in the regulation of stress-induced flowering in the short-day plant pharbitis (also called Japanese morning glory) Ipomoea nil (formerly Pharbitis nil) was studied. Pharbitis cv. Violet was induced to flower when grown in 1/100-strength mineral nutrient solution under non-inductive long-day conditions. All fully expanded true leaves were removed from seedlings, leaving only the cotyledons, and flowering was induced under poor-nutrition stress conditions. This indicates that cotyledons can play a role in the regulation of poor-nutrition stress-induced flowering. The expression of the pharbitis homolog of PHENYLALANINE AMMONIA-LYASE, the enzyme activity of phenylalanine ammonia-lyase (PAL; E.C. 4.3.1.5) and the content of SA in the cotyledons were all up-regulated by the stress treatment. The Violet was also induced to flower by low-temperature stress, DNA demethylation and short-day treatment. Low-temperature stress enhanced PAL activity, whereas non-stress factors such as DNA demethylation and short-day treatment decreased the activity. The PAL enzyme activity was also examined in another cultivar, Tendan, obtaining similar results to Violet. The exogenously applied SA did not induce flowering under non-stress conditions but did promote flowering under weak stress conditions in both cultivars. These results suggest that stress-induced flowering in pharbitis is induced, at least partly, by SA, and the synthesis of SA is promoted by PAL.
... A potential method to examine the role of phenylpropanoids in the growth and development of plants is to grow the plants in the presence of a metabolic inhibitor to prevent phenylpropanoid biosynthesis and examine the effects on growth and development. The most effective known PAL inhibitor is 2aminoindane-2-phosphonic acid (AIP), a structural analog of phenylalanine (Appert et al. 2003). This compound has a K i / K m of 0.0025 for the Zea mays PAL enzyme, and a K i /K m of 0.005 for the tyrosine deamination activity of PAL (Zoń and Amrhein 1992), indicating that it is a potent inhibitor of this enzyme and the isoforms present in Z. mays. ...
Article
The Poaceae includes some of the most important food, fiber, and bio-fuel crops. While there have been many studies investigating the function of phenylpropanoids in this family, most of our understanding is based on correlative data rather than experimental evidence. The current study was conducted to evaluate the roles of phenylpropanoids in the growth and development of Zea mays and to develop an experimental model for further investigations. Z. mays seedlings were grown in vitro with various concentrations of the competitive phenylalanine ammonia lyase inhibitor, 2-aminoindane-2-phosphonic acid (AIP). Ferulic acid, a downstream biosynthetic product, was added to determine if it could rescue the induced phenotypes. At lower concentrations of AIP, plants exhibited elongated roots and shoots, but at higher concentrations, growth was extremely stunted. At the cellular level, the epidermal cells of roots cultured with AIP exhibited a loss of intercellular adhesion and organization, and their cell walls were more readily degraded by enzymatic digestion. These characteristics were accompanied by significant reductions in primary cell wall autofluorescence, indicating that less ferulic acid and other phenolics were incorporated in the cell wall. The majority of these symptoms could be partially or entirely rescued by ferulic acid, providing further evidence that these differences were due to the inhibition of phenylpropanoid biosynthesis. This study provides experimental evidence supporting and expanding upon hypothesized functions of phenylpropanoids in the growth and development of Z. mays and provides an experimental system for further investigations in the Poaceae and other taxonomic groups.
... PAL catalyzes the conversion of phenylalanine to t-cinnamic acid, and SA is one of the metabolic intermediates derived from t-cinnamic acid. Aminooxyacetic acid (AOA) and l-2-aminooxy-3-phenylpropionic acid (AOPP), which function as PAL inhibitors (Kessmann et al., 1990;Appert et al., 2003), inhibited stress-induced flowering in P. nil and P. frutescens (Hatayama and Takeno, 2003;Wada et al., 2010a, b). In addition, the inhibitory effect of AOA and AOPP was negated by t-cinnamic acid, SA and benzoic acid (a precursor of SA) in P. nil under stress conditions (Hatayama and Takeno, 2003;Wada et al., 2010b). ...
Article
The short-day plant, Lemna paucicostata (synonym Lemna aequinoctialis), was induced to flower when cultured in tap water without any additional nutrition under non-inductive long-day conditions. Flowering occurred in all three of the tested strains, and strain 6746 was the most sensitive to the starvation stress conditions. For each strain, the stress-induced flowering response was weaker than that induced by short-day treatment, and the stress-induced flowering of strain 6746 was completely inhibited by aminooxyacetic acid and l-2-aminooxy-3-phenylpropionic acid, which are inhibitors of phenylalanine ammonia-lyase. Significantly higher amounts of endogenous salicylic acid (SA) were detected in the fronds that flowered under the poor-nutrition conditions than in the vegetative fronds cultured under nutrition conditions, and exogenously applied SA promoted the flowering response. The results indicate that endogenous SA plays a role in the regulation of stress-induced flowering.
... Resistance to cell wall degradation in woody plants has previously been attributed to the presence of hydroxycinnamic acids, synthesized through the phenylpropanoid pathway (Butt 1985). To address this problem in American elm, a new approach was developed by culturing source tissues in the presence of 2-aminoindane-2-phosphonic acid, an inhibitor of phenylalanine ammonia lyase (PAL) (Appert et al. 2003;Jones et al. 2012). It is hypothesized that by selectively inhibiting the PAL enzyme, the concentration of cell wallreinforcing compounds such as ferulic and coumaric acid are reduced, making the cell walls more amenable to enzymatic degradation . ...
Article
Full-text available
This study describes a protocol for regeneration of plants from cell suspension-derived protoplasts of American elm (Ulmus americana). Efficient protoplast isolation was achieved from a two-phase culture system through the incorporation of 100 μM 2-aminoindan-2-phosphonic acid, with a yield of approximately 2 × 10(6) protoplasts/ml packed cell volume. Isolated protoplasts failed to survive in liquid or alginate bead culture systems but initiated and continued to divide when embedded in low melting point agarose beads. Protoplast-derived callus proliferated and differentiated into shoot buds in response to 10 or 20 μM thidiazuron. Differentiated buds elongated and continued to proliferate on elm shoot medium supplemented with 3.0 μM GA3. The protoplast-derived shoots rooted and acclimatized to greenhouse conditions and continued to grow. This system provides the first protoplast-to-plant regeneration system for American elm and provides a framework for the development of protoplast fusion or genome editing technologies.
... Michaelis-Menten kinetics and no cooperative behavior (Appert et al., 1994;Appert et al., 2003). This mechanistic discrepancy between the native and recombinant enzymes was ascribed to the presence of heterotetrameric isoforms in the purified native enzymes or the occurrence of posttranslational modification, which may not occur in the bacterial expression system. ...
Article
Full-text available
Phenylpropanoid biosynthesis in plants engenders a vast variety of aromatic metabolites critically important for their growth, development, and environmental adaptation. Some of these aromatic compounds have high economic value. Phenylalanine ammonia-lyase (PAL) is the first committed enzyme in the pathway; it diverts the central flux of carbon from primary metabolism to the synthesis of myriad phenolics. Over the decades, many studies have shown that exquisite regulatory mechanisms at multiple levels control the transcription and the enzymatic activity of PALs. In this review, we present a current overview on our understanding of the complicated regulatory mechanisms governing PAL's activity; we particularly highlight recent progresses in unraveling its post-translational modifications, its metabolite feedback regulation, and its enzyme organization.
... PAL catalyzes the conversion of phenylalanine to t-cinnamic acid, and SA is derived from the latter (Yalpani et al., 1993). Aminooxyacetic acid (AOA) and l-2-aminooxy-3phenylpropionic acid (AOPP), which function as PAL inhibitors (Havir, 1981;Kessmann et al., 1990;Appert et al., 2003), inhibit stress-induced flowering, and this inhibitory effect is negated by t-cinnamic acid and SA in pharbitis (Hatayama and Takeno, 2003;Wada et al., 2010b). The increase in PAL activity and flowering inhibition by AOA have been reported in pharbitis under LD conditions (Hirai et al., 1995). ...
Article
The short-day plant pharbitis (also called Japanese morning glory), Ipomoea nil (formerly Pharbitis nil), was induced to flower by poor-nutrition stress. This stress-induced flowering was inhibited by aminooxyacetic acid (AOA), which is a known inhibitor of phenylalanine ammonia-lyase (PAL) and the synthesis of indole-3-acetic acid (IAA) and 1-aminocycropropane-1-carboxylic acid (ACC) and thus regulates endogenous levels of salicylic acid (SA), IAA and polyamine (PA). Stress treatment increased PAL activity in cotyledons, and AOA suppressed this increase. The observed PAL activity and flowering response correlate positively, indicating that AOA functions as a PAL inhibitor. The inhibition of stress-induced flowering by AOA was also overcome by IAA. An antiauxin, 4-chlorophenoxy isobutyric acid, inhibited stress-induced flowering. Both SA and IAA promoted flowering induced by stress. PA also promoted flowering, and the effective PA was found to be putrescine (Put). These results suggest that all of the pathways leading to the synthesis of SA, IAA and Put are responsive to the flowering inhibition by AOA and that these endogenous factors may be involved in the regulation of stress-induced flowering. However, as none of them induced flowering under non-stress conditions, they may function cooperatively to promote flowering.
... The activity of PAL can be inhibited by 2-aminoindane-2-phosphonic acid monohydrate (AIP) (Zoń and Amrhein 1992;Zoń et al. 2004), which is a competitive slow-binding inhibitor of PAL with reversible inhibition (Appert et al. 2003). In previous studies, AIP has been shown to inhibit the accumulation of various kinds of phenolic compounds (Zoń and Amrhein 1992;Reuber et al. 1993;Gitz et al. 1998;Julkunen-Tiitto 2000, 2003). ...
Article
Full-text available
We studied phenolic metabolism and plant growth in birch seedlings at the beginning of their development by inhibiting phenylalanine ammonia lyase (PAL), which is the first committed step in phenylpropanoid metabolism. Betula pubescens (Ehrh.) seeds were germinated in inhibitor-free media and the seedlings were transferred to hydroponic culture at the cotyledon stage. They were 6days old at the start of the experiment, which lasted for 3weeks. PAL activity was inhibited by three different concentrations of 2-aminoindane-2-phosphonic acid monohydrate (AIP) in the growing media. At the end of 3weeks, phenolics in all plant parts (roots, stem, cotyledons, first, second and third true leaves) were determined. AIP inhibited strongly the accumulation of phenolic acids, salidroside, rhododendrins, ellagitannins and their precursors, flavan-3-ols, and soluble condensed tannins. The accumulation of lignin and flavonol glycoside derivatives was moderately inhibited. The accumulation of flavonol glycosides, such as quercetin glycosides and kaempferol glycosides, was not generally inhibited, even in leaves that emerged during the experiment, while the accumulation of insoluble condensed tannins was inhibited only slightly and not in all plant parts. This suggests that flavonol glycosides, which may have a UV-B protective role, and insoluble condensed tannins, which may have structural functions, are prioritized in seedling development. Inhibition of PAL with AIP decreased seedling growth and possible reasons for this are discussed.
... To verify whether the exogenously applied allelochemicals are channeled via specific entry points of the phenylpropanoid pathway, we performed experiments using three selective enzyme inhibitors: AIP [24], PIP and MDCA [25]. AIP is a competitive inhibitor of PAL, PIP is an irreversible inhibitor of C4H and MCDA is a competitive inhibitor of 4CL ( Figure 8). ...
Article
Full-text available
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.
... These results suggest that the metabolic pathway mediated by PAL is involved in the regulation of stress-induced flowering. Aminooxyacetic acid (AOA) and L-2-aminooxy-3phenylpropionic acid, which are PAL inhibitors [19,20], inhibited stress-induced flowering in pharbitis [9,14]. PAL catalyzes the conversion of phenylalanine to t-cinnamic acid, from which anthocyanins, chlorogenic acid and SA are produced. ...
Article
Full-text available
Many plant species are induced to flower by stress. Stress-induced flowering has been studied mostly in the short-day plant pharbitis (also called Japanese morning glory; Ipomoea nil, formerly Pharbitis nil). In this article, physiological characteristics, the regulation by salicylic acid (SA) and the expression of flowering-related genes in stress-induced flowering in pharbitis are reviewed. Pharbitis flowered under long-days in response to poor nutrition or low temperature. The pharbitis plants induced to flower by stress reached anthesis, fruited and produced fertile seeds. The progeny of the stressed plants developed normally. Grafting experiments indicated that a transmissible flowering stimulus is involved in poor nutrition stress-induced flowering. Aminooxyacetic acid (AOA), a phenylalanine ammonia-lyase (PAL) inhibi-tor, inhibited the stress-induced flowering, and this inhibition was overcome by SA. Stress induced PAL activity and SA biosynthesis. PnFT2, a pharbitis ortholog of the flowering gene FLOWERING LOCUS T of Arabidopsis thaliana, was expressed when the plants were induced to flower by stress. The overexpression of PnFT2 induced flowering, and PnFT2RNAi inhibited it. AOA inhibited PnFT2 expression induced by stress, and SA eliminated this inhibitory effect. SA enhanced PnFT2 expression under poor nutrition but not under non-stressful conditions. Therefore, stress may induce the production of SA and other unknown factor(s) that may work in combination to induce PnFT2 expression and flowering.
... In addition, PAL is also involved in the production of salicylic acid (SA) (Verberne et al. 1999), which plays crucial roles in the activation of various plant defense responses such as host cell death and the containment of pathogen spread (Durner et al. 1997). To reduce synthesis of phenolic compounds in a variety of systems, AIP can be used both in vitro and in vivo as a competitive inhibitor of PAL (Appert et al. 2003;Bagheri et al. 2011). In this study, 2 µM of AIP was found able to effectively inhibit callus browning, significantly reduce EGC abundance in the treated plant materials, and greatly improve callus induction and growth. ...
Article
Explant oxidative browning and necrosis of Camellia sinensis var. sinensis is a severe problem in tissue culture, often associated with the exuded phenolic compounds and microbial contamination from the explants. In this study, 2-aminoindane- 2-phosphonic acid (AIP), an inhibitor of the polyphenol production-required enzyme phenylalanine ammonia lyase (PAL), and different antibiotics were tested to control tea explant necrosis and browning. These compounds were supplemented in the regular plant growth medium together with 6-benzylaminopurine and thidiazuron at different concentrations. Our data indicated that application of 2 μM of AIP was able to effectively inhibit callus browning, significantly reduce EGC abundance, and greatly improve callus induction and growth. Moreover, the use of 150 mg/L of timentin and 30 mg/L gentamycin resulted in an effective elimination of the surface and endophytic microbes associated with explants of C. sinensis var. sinensis. Our study revealed that the inhibition of PAL using AIP combined with the two tested antibiotics could open up new doors to control oxidative tissue browning and endophyte contamination in tissue culture for tea genetic manipulation.
... The effect of the addition of the PAL inhibitor AIP is more difficult to interpret as it clearly reduced, but did not eliminate, the induction of PR1 after H. parviporum infection (Figure 7b). AIP acts as reversible competitive inhibitor; thus, it is possible that a certain PAL activity that could convert phenylalanine to cinnamic acid remains after the treatment with AIP (Appert et al., 2003), and that this residual PAL activity produces sufficient cinnamic acid for some pathogen-induced SA production. Most likely, however, the JAmediated induction of PR1 in Norway spruce is not dependent on PALmediated production of cinnamic acid. ...
Thesis
Full-text available
Norway spruce [Picea abies (L.) Karst.] is a main tree species in European forests and is important both ecologically and economically. The root rot fungus Heterobasidion annosum sensu lato (s.l.) is the main P. abies pathogen. Including resistance in breeding programs would help mitigating the impact of the pathogen but knowledge regarding defense mechanisms in P. abies needs a better understanding. The work within this thesis intended to expand the existing knowledge on P. abies resistance mechanisms focusing on hormone signaling, flavonoid biosynthesis and its transcriptional regulation. I found that jasmonic acid is the major hormone controlling defense signaling pathways in P. abies. Furthermore, we validated a candidate gene, PaLAR3, as a resistance marker for H. annosum s.l. in P. abies. PaLAR3 encodes an enzyme responsible for the synthesis of (+) catechin, which showed a fungistatic effect on H. parviporum. Analysis of genetic diversity revealed two allelic lineages of PaLAR3 that showed significant differences in fungal resistance and (+) catechin content that were explained by dissimilarities in inducibility. We studied the role of PaNAC03, a transcription factor that is associated with H. annosum s.l. infection. PaNAC03 not only showed repression of multiple genes including PaLAR3, but bound only to the promoter of one of the PaLAR3 allelic lineages explaining at least partly the differences in allelic expression that were observed. Finally, we identified a full repertoire of members of a MYB/bHLH/WDR transcription factor complex in Norway spruce, which showed differences in protein interactions and expression patterns, and also in ability to control the expression of genes in the flavonoid biosynthetic pathway including PaLAR3.
... Beispiele von Stress-induzierten Phenylpropanoiden (Dixon & Paiva, 1995) Weil PAL als Schlüsselenzym in Pflanzen ubiquitär ist aber nicht in Tieren vorkommt, ist das Enzym außerdem ein ideales target für Herbizide. Dies führte zur Synthese vieler in vitro und in vivo Inhibitoren, die auch heute noch weiterentwickelt werden (Laber et al., 1986;Zon & Amrhein, 1992;Appert et al., 2003). ...
Article
Die Phenylalanin-Ammoniak-Lyase (PAL) aus Petroselinum crispum wurde rekombinant exprimiert, gereinigt und kristallisiert. Die Struktur wurde mit röntgenkristallographischen Methoden bis zu einer Auflösung von 1.7 Å gelöst und verfeinert. Das Enzym spielt eine Schlüsselrolle im sekundären Phenylpropanoidmetabolismus der Pflanzen. Die PAL ist ein fast ausschließlich alpha-helicales Protein mit 23 teilweise sehr langen alpha-Helices und acht kurzen beta-Faltblattsträngen. Ein PAL-Protomer besteht aus 716 Aminosäureresten und kann in eine MIO-Domäne, eine core-Domäne und eine shielding-Domäne unterteilt werden. In der Quartärstruktur lagern sich jeweils vier Protomere zu einem D2 Tetramer zusammen. Aus struktureller Sicht kann das homologe Enzym Histidin-Ammoniak-Lyase (HAL) als ein Vorgänger der PAL angesehen werden. Der Hauptunterschiede zwischen der um 207 Aminosäurereste längeren PAL und der HAL ist die Addition einer shielding-Domäne, die den Zugang zum aktiven Zentrum der PAL erschwert und somit mögliche Angriffe von Nucleophilen verhindert. Dies könnte dem Enzym helfen, in gestresstem Pflanzengewebe zu arbeiten. PAL katalysiert die nichtoxidative Eliminierung von Ammoniak aus L Phenylalanin, wobei trans-Zimtsäure entsteht. Diese ungewöhnliche Reaktion erfordert die Existenz einer elektrophilen Gruppe im Enzym. Es konnte gezeigt werden, dass diese elektrophile Gruppe bei der PAL ein durch eine posttranslationale, autokatalytische Zyklisierung des Proteinrückgrats gebildetes 4 Methyliden-imidazol 5 on (MIO) ist. Das MIO war erstmals bei der HAL entdeckt worden und ist, was Bildung und molekulare Struktur betrifft, mit dem Chromophor des Grün-Fluoreszierenden-Proteins (GFP) verwandt. Im Falle der vorliegenden Struktur der PAL wurde das MIO als DTT-Addukt erhalten, was auch durch Aktivitätsmesssungen und massenspektrometrische Untersuchungen bestätigt wurde. Es wurde ein Reaktionsmechanismus postuliert, der sich auf die gelöste Struktur, publizierte Mutationsstudien und Aktivitätsmessungen stützt. Durch Vergleiche auf den Ebenen der Raumstruktur sowie der Aminosäuresequenz konnte die Existenz einer PAL-HAL-Superfamilie gezeigt werden. In einem weiteren Teil der Arbeit konnte für die Tyrosin-Aminomutase (TAM) aus Jaspis splendens ein Expression- und Reinigungssystem etabliert werden. Es konnten außerdem Bedingungen zur Kristallisation des Enzyms gefunden werden, wobei die Qualität der erhaltenen Kristalle bislang allerdings noch keine Röntgenstrukturanalyse ermöglicht. Außerdem wurde im Rahmen dieser Arbeit die Ecto-ADP-Ribosyltransferase 2.2 (ART2.2) aus Rattus norvegicus untersucht. Wichtigstes Ziel war hier die Untersuchung der enzymatischen Aktivität. Mit einem neu etablierten RP HPLC-Test zur Messung der NAD+-Hydrolyseaktivität konnte die spezifische Aktivität des ART2.2 WT zu 26 U/mg und seine kinetischen Parameter zu kcat=12/s und KM=0.03 mM bestimmt werden, diese Werte liegen im üblichen Bereich von Hydrolasen. Da bislang kein exogenes Zielprotein der ART2.2 bekannt ist, deuten die gemessenen Werte darauf hin, dass die physiologische Rolle der ART2.2 die Hydrolyse von extrazellulärem NAD+ ist. Um die Rolle wichtiger Aminosäurereste bei der Katalyse zu untersuchen, wurden verschiedene Mutanten generiert und deren Aktivität ebenfalls mit Hilfe des RP HPLC-Tests gemessen, wobei die Wichtigkeit des in der ART-Familie konservierten Aminosäurerests E189 für die Katalyse belegt werden konnte. Zur Untersuchung von Substratbindung und Katalysemechanismus konnten durch Tränkexperimente von Kristallen und anschließender Strukturlösung mit der Methode des molekularen Ersatzes drei Komplexstrukturen erhalten werden. Die des Wildtyp im Komplex mit dem Inhibitor TAD bei 2.0 Å, sowie Strukturen der Mutanten E189I und E189A im Komplex mit dem physiologischen Substrat NAD+ beziehungsweise mit dem Substratanalogon NADH bei jeweils 2.6 Å. Das Substrat wird in einer tiefen Spalte, die sich fast über die ganze Breite des Enzym hinzieht, gebunden. Vor dem Hintergrund der Komplexstrukturen wurde außerdem ein Mechanismus für NAD+-Hydrolyse und Auto ADP-Ribosylierung der ART2.2 postuliert. Für beide Mechanismen ist das unter den ARTs konservierte R S QxE-Motiv essentiell, das im Falle der ART2.2 aus den Aminosäureresten R126, S147, Q187 und E189 besteht. Auch der Vorschlag, dass die Auto ADP-Ribosylierung bei der ART2.2 durch Anheftung der ADP-Riboseeinheit an den Aminosäurerest R184 geschieht, wurde strukturell untermauert.
... To determine whether formation of benzaldehyde from Phe occurs via CA, we conducted experiments with a specific inhibitor of PAL, 2-aminoindanephosphonate (AIP; Zon and Amrhein, 1992;Appert et al., 2003). In these experiments, excised corolla limbs were pretreated with AIP (25 mmol) for 40 min and then floral scent was collected for 4 h from limbs continuously fed with 2 H 5 -Phe. ...
Article
Full-text available
In vivo stable isotope labeling and computer-assisted metabolic flux analysis were used to investigate the metabolic pathways in petunia (Petunia hybrida) cv Mitchell leading from Phe to benzenoid compounds, a process that requires the shortening of the side chain by a C(2) unit. Deuterium-labeled Phe ((2)H(5)-Phe) was supplied to excised petunia petals. The intracellular pools of benzenoid/phenylpropanoid-related compounds (intermediates and end products) as well as volatile end products within the floral bouquet were analyzed for pool sizes and labeling kinetics by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Modeling of the benzenoid network revealed that both the CoA-dependent, beta-oxidative and CoA-independent, non-beta-oxidative pathways contribute to the formation of benzenoid compounds in petunia flowers. The flux through the CoA-independent, non-beta-oxidative pathway with benzaldehyde as a key intermediate was estimated to be about 2 times higher than the flux through the CoA-dependent, beta-oxidative pathway. Modeling of (2)H(5)-Phe labeling data predicted that in addition to benzaldehyde, benzylbenzoate is an intermediate between l-Phe and benzoic acid. Benzylbenzoate is the result of benzoylation of benzyl alcohol, for which activity was detected in petunia petals. A cDNA encoding a benzoyl-CoA:benzyl alcohol/phenylethanol benzoyltransferase was isolated from petunia cv Mitchell using a functional genomic approach. Biochemical characterization of a purified recombinant benzoyl-CoA:benzyl alcohol/phenylethanol benzoyltransferase protein showed that it can produce benzylbenzoate and phenylethyl benzoate, both present in petunia corollas, with similar catalytic efficiencies.
... The enzyme is accumulated in the vicinity of the affected tissue (Mauch-Mani and Slusarenko, 1996; Ehness et al., 1997). Because PAL is ubiquitously distributed in plants and absent in animals, it is a promising target for herbicides; various inhibitors are currently being developed (Laber et al., 1986; Appert et al., 2003). ...
Article
Because of its key role in secondary phenylpropanoid metabolism, Phe ammonia-lyase is one of the most extensively studied plant enzymes. To provide a basis for detailed structure-function studies, the enzyme from parsley (Petroselinum crispum) was crystallized, and the structure was elucidated at 1.7-A resolution. It contains the unusual electrophilic 4-methylidene-imidazole-5-one group, which is derived from a tripeptide segment in two autocatalytic dehydration reactions. The enzyme resembles His ammonia-lyase from the general His degradation pathway but contains 207 additional residues, mainly in an N-terminal extension rigidifying a domain interface and in an inserted alpha-helical domain restricting the access to the active center. Presumably, Phe ammonia-lyase developed from His ammonia-lyase when fungi and plants diverged from the other kingdoms. A pathway of the catalyzed reaction is proposed in agreement with established biochemical data. The inactivation of the enzyme by a nucleophile is described in detail.
... In addition, PAL is also involved in the production of salicylic acid (SA) (Verberne et al. 1999), which plays crucial roles in the activation of various plant defense responses such as host cell death and the containment of pathogen spread (Durner et al. 1997). To reduce synthesis of phenolic compounds in a variety of systems, AIP can be used both in vitro and in vivo as a competitive inhibitor of PAL (Appert et al. 2003;Bagheri et al. 2011). In this study, 2 µM of AIP was found able to effectively inhibit callus browning, significantly reduce EGC abundance in the treated plant materials, and greatly improve callus induction and growth. ...
Article
Full-text available
Explant oxidative browning and necrosis of Camellia sinensis var. sinensis is a severe problem in tissue culture, often associated with the exuded phenolic compounds and microbial contamination from the explants. In this study, 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of the polyphenol production-required enzyme phenylalanine ammonia lyase (PAL), and different antibiotics were tested to control tea explant necrosis and browning. These compounds were supplemented in the regular plant growth medium together with 6-benzylaminopurine and thidiazuron at different concentrations. Our data indicated that application of 2 µM of AIP was able to effectively inhibit callus browning, significantly reduce EGC abundance, and greatly improve callus induction and growth. Moreover, the use of 150 mg/L of timentin and 30 mg/L gentamycin resulted in an effective elimination of the surface and endophytic microbes associated with explants of C. sinensis var. sinensis. Our study revealed that the inhibition of PAL using AIP combined with the two tested antibiotics could open up new doors to control oxidative tissue browning and endophyte contamination in tissue culture for tea genetic manipulation.
... When PVP was added to the PVPP culture of guar cotyledon protoplasts, not only was it found to enhance the necrosis inhibition, but it also improved the protoplast division frequency. Another compound known to decrease tissue browning is 2-aminoindane-2-phosphonic acid (AIP), which is a reversible inhibitor of phenylalanine ammonia lyase (PAL), an enzyme necessary for polyphenol production (Appert et al., 2003). While the inhibition of PAL was able to increase the cell wall digestibility and facilitate sustained cell division in American elm (Ulmus americana), extended inhibition results in decreased shoot growth in tissue culture (Jones et al., 2012). ...
Article
Full-text available
The development of gene-editing technology holds tremendous potential for accelerating crop trait improvement to help us address the need to feed a growing global population. However, the delivery and access of gene-editing tools to the host genome and subsequent recovery of successfully edited plants form significant bottlenecks in the application of new plant breeding technologies. Moreover, the methods most suited to achieve a desired outcome vary substantially, depending on species' genotype and the targeted genetic changes. Hence, it is of importance to develop and improve multiple strategies for delivery and regeneration in order to be able to approach each application from various angles. The use of transient transformation and regeneration of plant protoplasts is one such strategy that carries unique advantages and challenges. Here, we will discuss the use of protoplast regeneration in the application of new plant breeding technologies and review pertinent literature on successful protoplast regeneration.
Chapter
The biological activity and natural occurrence of the aminophosphonic acids were described half a century ago. Since then the chemistry and biology of this class of compounds have developed into the separate field of phosphorus chemistry. Today it is well acknowledged that these compounds possess a wide variety of promising, and in some cases commercially useful, physiological activities. Thus, they have found applications ranging from agrochemical (with the herbicides glyphosate and bialaphos being the most prominent examples) to medicinal (with the potent antihypertensive fosinopril and antiosteoporetic bisphosphonates being examples). Key WordsAminophosphonates-Bisphosphonates-Drug design and development-Enzyme inhibitors-Phosphono peptides-Structural analogues-Transition state analogues
Article
INF1 elicitin, a proteinaceous elicitor produced by Phytophthora infestans, induces a hypersensitive response in tobacco BY-2 cells. In response to elicitin, tobacco cells produce both reactive oxygen species (ROS) and ethylene (ET). To investigate the regulation of elicitin-induced ET production, we pharmacologically analyzed the effects of several chemicals on ET production. Inhibitors of ROS generation or ROS chelators efficiently inhibited ET production, whereas simultaneous treatment of a superoxide anion-generating system with salicylhydroxamic acid recovered ET production. In an in vitro experiment, superoxide anion was necessary and sufficient for conversion of 1-aminocyclopropane-1-carboxylate (ACC) to ET because ET was produced from ACC solely in the presence of the superoxide-generating chemical KO2. ET production was also inhibited by lipoxygenase (LOX) inhibitors, indicating a possible involvement of LOX-mediated generation of superoxide anion and ET production itself. Furthermore, elicitin-induced ET production was completely inhibited by the protein synthesis inhibitor cycloheximide but recovered after exogenous application of ACC, indicating that de novo protein synthesis is required for ACC accumulation, leading to ET production. We also investigated the effects of several phytohormones on elicitor-induced ET production and discuss their role in the defense response.
Article
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.
Article
The effects of 2-aminoindane-2-phosphonic acid (AIP), a potent phenylalanine ammonia-lyase (PAL) inhibitor, on the accumulation of cadmium and nickel in chamomile (Matricaria chamomilla) were examined in this study. In vitro assay of AIP effect showed a 90% reduction in PAL activity. In plants cultured for 7 days in Cd or Ni solutions with AIP, PAL activity was higher in both shoots and roots (in comparison with metals without AIP), and was correlated with changes in free phenylalanine content. Individual amino acids were both positively and negatively affected by AIP, with the accumulation of tyrosine and proline showing increases in some variants. Contents of soluble phenols and flavonoids were not considerably affected, while amounts of coumarin-related compounds, cell wall-bound phenols and phenolic acids were substantially reduced in AIP-treated variants. Lignin accumulation decreased in controls and increased in Cd variants in response to AIP. Shoot Cd content was depleted, but shoot Ni was elevated by AIP. Total root content of Cd and Ni decreased in +AIP variants. AIP also caused more expressive changes in hydrogen peroxide and superoxide content in Cd than in Ni variants. Our results indicate that phenols have important roles in the uptake of Cd and Ni. The present findings are discussed in the context of available data regarding AIP's effect on phenols.
Article
The structure of 2-aminoindane-2-phosphonic acid (AIP) was studied using X-ray crystallography, NMR spectroscopy, molecular modelling, IR spectroscopy, and potentiometric titration. In the solid state, a thermodynamically stable conformer of AIP is one in which the phosphonic group occupies the equatorial position and the amino group the axial position. The NMR data suggest that fast equilibrium in solution between the two conformers of AIP is significantly shifted toward the equatorial conformer (EC). Both solid state studies, that is X-ray analysis and IR spectroscopy of AIP, revealed the presence of hydrogen-bonded water. Ab initio calculations in the gas phase indicate only a small barrier between the two possible conformations of AIP. Binding studies of both conformers, in various protonation states, to the model of the phenylalanine ammonia-lyase structure suggest that only the axial phosphonic group conformer is docked specifically. Indications from modelling are that phenylalanine ammonia-lyase binds AIP's conformer with higher specificity and that the molecular reorganisation required can be responsible for the experimentally observed time-dependent inhibition.
Article
The effect of phenylalanine ammonia-lyase (PAL) inhibition by 2-aminoindane-2-phosphonic acid (AIP) in copper-exposed diploid and tetraploid chamomile (Matricaria chamomilla ) roots has been studied in a short-term experiment (24 h). Cu evoked stronger induction of PAL activity and accumulation of soluble phenols, flavonols (quercetin and kaempferol), and lignin in diploid plants, whereas AlCl3-reactive flavonoids and phenolic acids did not differ with respect to ploidy. Amounts of hydrogen peroxide and superoxide also preferentially increased in diploid. Surprisingly, PAL activity was restored in both +AIP cultivars, being inversely correlated with the accumulation of free phenylalanine. Notwithstanding this, total soluble phenols and flavonols were more depleted in Cu+AIP diploid roots. Soluble proteins decreased in response to Cu, and AIP had no effect. Among free amino acids, proline increased more visibly in Cu+AIP diploid, suggesting that this could be a protective mechanism in conditions with depleted content of phenols. Decrease in potassium content was ploidy-independent, calcium increased in all Cu variants, and Fe increased in Cu-exposed tetraploid. Shoot Cu content did not differ in Cu-exposed cultivars, but diploid roots contained more Cu. AIP decreased root Cu but increased shoot Cu amounts in diploid, whereas tetraploid plants did not exhibit similar responses. These data indicate that inhibition of root phenolic metabolism by AIP was effective enough, allowing Cu to accumulate in diploid shoots. The present findings are discussed in the context of available data about AIP effects and with respect to the role of phenols in metal uptake.
Article
Acetophenone (AP) and 1-phenylethanol (1PE) are the two major endogenous volatile compounds in flowers of Camellia sinensis var. Yabukita. Until now no information has been available on the biosynthesis of AP and 1PE in plants. Here we propose that AP and 1PE are derived from L-phenylalanine (L-Phe), based on feeding experiments using stable isotope-labeled precursors L-[(2)H(8)]Phe and L-[(13)C(9)]Phe. The subacid conditions in the flowers result in more hydrogenation than dehydrogenation in the transformation between AP and 1PE. Due to the action of some enzyme(s) responsible for the formation of (R)-1PE from AP in the flowers, (R)-1PE is the dominant endogenous steroisomer of 1PE. The modification of 1PE into nonvolatile glycosidic forms is one of the reasons for why only a little 1PE is released from the flowers. The levels of AP, 1PE, and glycosides of 1PE increase during floral development, whereas the level of L-Phe decreases. These metabolites occur mostly in the anthers.
Article
Full-text available
Phosphonylated azaheterocycles are an important class of compounds with high biological potential as conformationally restricted bioisosteres of amino acids. Therefore, it is of interest to synthesize conformationally constrained amino phosphonates. We wanted to investigate possible routes via ring opening of alpha-amino phosphonates with an oxanorbornene skeleton, as these can be synthesized with high stereoselectivity. This was achieved using different Lewis acids, leading to a range of products. The reaction with TiCl(4) and FeCl(3) was modelled at a DFT level of theory to get insight in the pathways towards the corresponding products. To ease the work up, the Fe(iii) catalyst was coated on montmorillonite clay, but this accelerated aromatization after ring opening. Quenching the FeCl(3) catalyzed reaction mixture on celite caused complete aromatization.
Article
The committed biosynthetic reaction to benzoyl-coenzyme A in the marine bacterium “Streptomyces maritimus” is carried out by the novel prokaryotic phenylalanine ammonia lyase (PAL) EncP, which converts the primary amino acid l-phenylalanine to trans-cinnamic acid. Recombinant EncP is specific for l-phenylalanine and shares many biochemical features with eukaryotic PALs, which are substantially larger proteins by ∼200 amino acid residues.
Article
Despite the pharmaceutical importance of hypericin as a drug lead molecule in various medical applications, the hypericin biosynthesis pathway is not known. Herein, we report on the ability to reversibly inhibit hypericin biosynthesis via impairing the dark glands that synthesize/accumulate hypericin, and reveal the correlation between important genes and hypericin biosynthesis. The number and size of dark glands were significantly reduced in shoots regenerated from St. John’s wort (Hypericum perforatum L.) root explants on medium containing sublethal level (5 µM) of glyphosate (N-(Phosphonomethyl)glycine). Ultra-Performance liquid chromatography (UPLC) system coupled with photodiode array (PDA) and fluorescence (FLR) analysis revealed that naphthodianthrones and their putative precursors i.e., emodin and bisanthrones were significantly inhibited in the presence of glyphosate, and these compounds could be fully recovered by withdrawing glyphosate, whereas phenolic compounds and phloroglucinols remain mostly unaffected. The expression of polyketide synthases (HpPKS2 and HpOKS) and phenolic oxidative coupling proteins (HpPOCP2, HpPOCP1 and HpPOCP3) were down-regulated upon glyphosate treatment and up-regulated after glyphosate withdrawal, highlighting the link between these genes and hypericin biosynthesis.
Chapter
Computer-aided techniques of rational design of enzyme inhibitors were reviewed. In silico lead generation and optimization protocols were outlined and several methods of inhibitor potency estimation by both empirical scoring functions as well as ab initio based calculations were described. Two representative examples of successful computer-aided analysis and design of novel, highly potent inhibitors of leucine aminopeptidase and glutamine synthetase were demonstrated. In addition fully nonempirical and systematic analysis of the physical nature of enzyme active site interactions has been performed for series of leucine aminopeptidase (LAP) and phenylalanine ammonia lyase (PAL) inhibitors. Results derived from ab initio calculations indicate that inhibitory activity is controlled by interactions with limited number of active site residues. Examination of entire hierarchy of theoretical models indicates that the inhibitory activity could be well represented by electrostatic interactions, leading to so called ‘‘electrostatic key-lock’’ principle
Article
Full-text available
A model of a continuous L-phenylalanine ammonia-lyase (PAL) fermentation process by Aureobasidium pullulans (Y863) pigment yeast strain was developed. As a basis of the kinetic model, differential equations of Luedeking-Piret model combined with Monod were used. It was found that the kinetic growth of Aureobasidium pullulans (Y863) and synthesis of PAL were carried out not only in case of decrease of phenylalanine content in the substrate, but also in the course of biosynthesis of the desired product. As an effective criterion of the biokinetics' selected performance, the PAL target product was selected, determined by the quantity of the substrate stream and the concentration of the target product in the culture medium. The obtained dependencies of the phenylalanine concentration in the nutrient solution for every fixed value of the feed stream have a unified dynamics of change. The obtained kinetic dependences can be used for both qualitative and quantitative characteristics of the process in the development of PAL production technology.
Article
Full-text available
Benzocyclobutene and its derivatives are a significant class of compounds useful in biological and polymeric science. In this review, we summarize the synthesis of BCBs, and its utilization to construct diverse bioactive natural products, medicinally interesting molecules, functional and polymeric materials. Abstract The benzocyclobutene (BCB) moiety is present in several bioactive natural products and medicinally interesting molecules. BCB is also a valuable synthon for building a variety of polymers by the virtue of its propensity to undergo polymerization. The research on BCB chemistry has grown in the last few decades due to its unique applications in various fields of science. In this review, we have included various methods for the preparation of BCBs and highlighted their applications in the construction of diverse complex structural frameworks. We believe that this review will be useful to synthetic chemists and polymer scientists, and we anticipate several new applications will emerge in this promising area.
Article
Full-text available
Reacting to environmental exposure, most higher plants activate secondary metabolic pathways, such as the metabolism of phenylpropanoids. This pathway results in the formation of lignin, one of the most important polymers of the plant cell, as well as a wide range of phenolic secondary metabolites. Aromatic amino acids, such as phenylalanine and tyrosine, largely stimulate this process, determining two ways of lignification in plant tissues, varying in their efficiency. The current study analyzed the effect of phenylalanine and tyrosine, involved in plant metabolism through the phenylalanine ammonia-lyase (PAL) pathway, on the synthesis and accumulation of phenolic compounds, as well as lignin by means of the expression of a number of genes responsible for its biosynthesis, based on the example of common wheat (Triticum aestivum L.).
Article
Full-text available
A series of phosphonic acid analogues of phenylglycine variously substituted in phenyl ring have been synthesized and evaluated for their inhibitory activity towards potato L-phenylalanine ammonia lyase. Most of the compounds appeared to act as moderate (micromolar) inhibitors of the enzyme. Analysis of their binding performed using molecular modeling have shown that they might be bound either in active site of the enzyme or in the non-physiologic site. The latter one is located in adjoining deep site nearby the to the entrance channel for substrate into active site.
Article
Full-text available
Aromatic amino acid ammonia-lyases and aromatic amino acid 2,3-aminomutases contain the post-translationally formed prosthetic 3,5-dihydro-4-methylidene-5H-imidazol-5-one (MIO) group. MIO-enzymes catalyze the stereoselective synthesis of α- or β-amino acid enantiomers, making these chemical processes environmentally friendly and affordable. Characterization of novel inhibitors enables structural understanding of enzyme mechanism and recognize promising herbicide candidates as well. The present study found, that both enantiomers of the aminophosphonic acid analogue of the natural substrate phenylalanine and a novel derivative bearing a methylidene at the β-position inhibited phenylalanine ammonia-lyases (PAL), representing MIO enzymes. X-ray methods unambiguously determined the absolute configuration of all tested enantiomers during their synthesis. Enzyme kinetic measurements revealed the enantiomer of the methylidene substituted substrate analogue being mirror image relation to the natural L-phenylalanine as the strongest inhibitor. Isothermal titration calorimetry (ITC) confirmed the binding constants and provided a detailed analysis of the thermodynamic driving forces of ligand binding. Molecular docking suggested that binding of the (R)- and (S)-enantiomers is possible by a mirror image packing.
Article
Five new geminal aminocycloalkanephosphonic acids (4-8) containing both an aromatic ring and a cycloalkane ring were synthesized and evaluated as potential inhibitors of buckwheat phenylalanine ammonia-lyase (PAL). Within the set of compounds which are related to 2-aminoindane-2-phosphonic acid (AIP, 3), a known powerful inhibitor of PAL, racemic 1-aminobenzocyclobutene-1-phosphonic acid (4), was six times weaker than AIP as an in vitro inhibitor of buckwheat PAL, but six times stronger than AIP as an in vivo inhibitor of phenylalanine-derived anthocyanin synthesis in buckwheat. This article is protected by copyright. All rights reserved.
Article
A variety of nitriles was reduced to diisobutylaluminum salts of aldimines, to which diisopropyl phosphite was added. The corresponding 1-aminophosphonates were either deprotected to give racemic 1-aminophosphonic acids or reacted with Boc2O to yield N-Boc-protected 1-aminophosphonates. The enantiomers of 2-benzylthio-1-(t-butoxycarbonylamino)propylphosphonate were obtained from the racemate by chiral HPLC and converted to phosphonic acid analogs of (R)- and (S)-homocysteine, (R)- and (S)-2-aminobutyric acid and (S)-methionine, all of ee >97% as determined by chiral HPLC.
Chapter
Plants have a tendency to flower under unsuitable growth conditions. Stress factors, such as poor nutrition, high or low temperature, high- or low-intensity light, and ultraviolet light, have been implicated in this stress-induced flowering. The stressed plants do not wait for the arrival of a season when photoperiodic conditions are suitable for flowering, and such precocious flowering might assist in species preservation. Stress-induced flowering has been well studied in Pharbitis nil (synonym Ipomoea nil), Perilla frutescens var. crispa, Lemna paucicostata (synonym Lemna aequinoctialis) and Arabidopsis thaliana. The phenylalanine ammonia-lyase (PAL) inhibitor suppresses stress-induced flowering in P. nil, and this effect was reversed with salicylic acid (SA). The PAL gene expression, PAL enzyme activity and SA content in the cotyledons increased during stress-induced flowering. These results suggest that SA mediates stress-induced flowering.
Article
Several carboxylic, phosphonic, phosphinic, boronic and nitro analogues of (E)-cinnamic acid were synthesized. These and other compounds related to (E)-cinnamic acid were evaluated as potential inhibitors of both phenylalanine ammonia-lyase and of anthocyanin biosynthesis in buckwheat. The most potent inhibition was found for 3-phenylprop-2-ynoic acid (21), however its K i is comparable to KM. The molecular modelling of the interaction of (E)-cinnamic acid (1) and 21 with PAL model suggests some similarities in the binding mode of both compounds.
Article
The benzocyclobutene-based alpha-amino acid derivative, ethyl 5-acetamido-2,4,5,6-tetrahydro-1H-cyclobuta[f] indene-5-carboxylate is synthesized via coupling of a benzocyclobutene-derived dibromide with ethyl isocyanoacetate as the key step, followed by hydrolysis and subsequent acetylation. This methodology is generalized in order to prepare various linearly and angularly fused indane-based alpha-amino acid derivatives.
Article
Full-text available
A one-step strategy to prepare (±)-diethyl 2-alkyl- and 2-aryl-(3-oxoisoindolin-1-yl)phosphonates is reported via a modified Kabachnik-Fields condensation of 2-carboxybenzaldehyde with an amine and triethyl phosphite using OSU-6, an MCM-41 type mesoporous hexagonal silica, as the catalyst. The reaction proceeds to form the target compounds in high yields with minimal purification requirements. The method is successful for benzylic, aromatic and aliphatic amines. An additional advantage of the current method is that the catalyst can be recycled up to four times with useful activity.
Article
Full-text available
Flavonoids and phenolic acids make up one of the most pervasive groups of plant phenolics. Due to their importance in plants and human health, it would be useful to have a better understanding of flavonoid concentration and biological activities that could indicate their potentials as therapeutic agents, and also for predicting and controlling the quality of medicinal herbs. Plants and herbs consumed by humans may contain thousands of different phenolic acid and flavonoid components. The effect of dietary phenolics is currently of great interest due to their antioxidative and possible anticarcinogenic activities. Phenolic acids and flavonoids also function as reducing agents, free radical scavengers, and quenchers of singlet oxygen formation. In addition, flavonoids and phenolic acids components play important roles in the control of cancer and other human diseases.
Article
A method for preparation of small, drug-like molecules based on indane, tetraline and isoquinoline scaffolds is developed starting from 1,2-bis(bromomethyl)tetrahalobenzenes using Diels—Alder reactions, alkylations and Suzuki couplings.
Article
2‐aminoindan‐2‐phosphonic acid (AIP) functions as a particular inhibitor of polyphenol biosynthesis in phenylpropanoid pathway. In this study, the role of AIP in browning resistance of fresh‐cut lily bulbs during storage was investigated. Application of 0.5 mmol/L AIP decreased browning degree of fresh‐cut lily bulbs, which was accompanied by reduced total phenolics content (TPC) and enzymes related to enzymatic browning including polyphenol oxidase (PPO), phenylalanine ammonia‐lyase (PAL) and peroxidase (POD). In comparison with the control, AIP efficiently maintained cell membrane integrity and lowered oxidative stress via inhibiting lipoxygenase (LOX) activity and malondialdehyde (MDA) content as well as promoting the activities of superoxide dismutase (SOD), catalase (CAT) and energy‐metabolism related enzymes, thus controlling browning of fresh‐cut lily bulbs. The browning inhibition of AIP was highly correlated with cell membrane structure, energy metabolism and enzymatic browning. These results show that AIP is a potential way to prevent browning of fresh‐cut lily bulbs.
Article
Anthraquinones (AQs) are compounds with a wide variety of interesting properties, which could be obtained by Rubia tinctorum suspension cultures. The present paper reports the effect of proline and aminoindan-2-phosphonic acid (AIP) on AQs production. The addition of proline (0.25mM) or AIP (100μM) enhance, up to 50% the AQ biosynthesis in R. tinctorum suspension culture. This increase is similar to those obtained by elicitation. Phenylalanine ammonia liase (PAL) activity was higher in presence of proline but no change in phenolic acid contents was found. Glucose-6-phosphate dehydrogenase (G6PDH) activity was not affected in cell cultures treated with proline. Addition of AIP produced PAL inhibition and lower levels of phenolic acid contents. These results show that it is possible to increase the AQs level in R. tinctorum suspension culture by metabolic manipulation and by inhibition of the key enzymes of the metabolic pathways that compete with AQs for a common substrate.
Article
Six derivatives of 2-aminoindane-2-phosphonic acid and 1-aminobenzylphosphonic acid were synthesized. The compounds were tested both as inhibitors of buckwheat phenylalanine ammonia-lyase (in vitro) and as inhibitors of anthocyanin biosynthesis (in vivo). (+/-)-2-Amino-4-bromoindane-2-phosphonic acid was found to be the strongest inhibitor from investigated compounds. The results obtained are a basis for design of phenylalanine ammonia-lyase inhibitors useful in the enzyme structure studies in photo labelling experiments.
Article
Full-text available
The phosphonic analogue of L-phenylalanine, (R)-(1-amino-2-phenylethyl)phosphonic acid (APEP), inhibits buckwheat phenylalanine ammonia-lyase (PAL) competitively with a Ki value of 1.5 μm. The Ki value for the (S)-enantiomer is 11.6 μm. The corresponding values for the enantiomers of the phosphonous analogue are 35 and 205 μm, respectively. APEP inhibits the light-induced synthesis of anthocyanin in hypocotyls of etiolated buckwheat seedlings and causes a specific increase in the endogenous phenylalanine concentration in buckwheat hypocotyls as well as other plant tissues. Kohlrabi seedlings develop normally in the presence of APEP, while their anthocyanin content is greatly reduced. These results indicate that APEP inhibits PAL in vivo. © 1986, Verlag der Zeitschrift für Naturforschung. All rights reserved.
Article
Full-text available
ʟ-α-Aminooxy-β-phenylpropionic acid (AOPP), a potent competitive inhibitor of phenylalanine ammonia-lyase (PAL), blocked light-induced phenylpropanoid synthesis in excised buckwheat hypocotyls and produced an up to 40-fold increase in the endogenous phenylalanine concentra­tion, while the level of all other amino acids was hardly affected. After a 24 h incubation in the light in the presence of 0.3 or 1 mᴍ AOPP phenylalanine alone constituted about 25% of the total soluble amino acids, compared to appr. 1% in the controls. In the presence of AOPP illuminated hypocotyls accumulated nearly 3 times more phenylala­nine than hypocotyls kept in the dark, indicating an enhancing effect of light on the flow of carbon through the shikimate pathway. Exogenously added [ ¹⁴ C] phenylalanine was extensively metab­olized by control tissue, but accumulated in AOPP treated tissue. In the presence of AOPP radio­activity from [ ¹⁴ C] shikimate accumulated predominantly in phenylalanine, and the flow of shi­kimate into tyrosine and phenylalanine was not affected by the inhibitor. Therefore, under these conditions no feedback control of phenylalanine and tyrosine synthesis from shikimate is apparent in buckwheat hypocotyls.
Article
Full-text available
When an enzyme exhibits a high affinity for an inhibitor, the steady-state analysis of the mechanism is complicated by the non-linearity of normal dose-response plots or of reciprocal replots. It is shown here that dose-response measurements generate a linear plot of inhibitor concentration divided by degree of inhibition against velocity without inhibitor divided by velocity with inhibitor; the concentration of enzyme may be derived from the extrapolated intercept of such plots, and the mechanism of inhibition from replots of the variation of the slope with substrate concentration. The limiting cases where virtually all inhibitor molecules are bound or virtually all are free are described, together with the situation when a significant proportion of the substrate becomes bound. This type of analysis indicates that the inhibitors of oxidative phosphorylation, rutamycin and bongkrekic acid, are tightly bound to rat liver mitochondria.
Article
Full-text available
The plant enzyme phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) shows homology to histidine ammonia-lyase (HAL) whose structure has been solved by X-ray crystallography. Based on amino-acid sequence alignment of the two enzymes, mutagenesis was performed on amino-acid residues that were identical or similar to the active site residues in HAL to gain insight into the importance of this residues in PAL for substrate binding or catalysis. We mutated the following amino-acid residues: S203, R354, Y110, Y351, N260, Q348, F400, Q488 and L138. Determination of the kinetic constants of the overexpressed and purified enzymes revealed that mutagenesis led in each case to diminished activity. Mutants S203A, R354A and Y351F showed a decrease in kcat by factors of 435, 130 and 235, respectively. Mutants F400A, Q488A and L138H showed a 345-, 615- and 14-fold lower kcat, respectively. The greatest loss of activity occurred in the PAL mutants N260A, Q348A and Y110F, which were 2700, 2370 and 75 000 times less active than wild-type PAL. To elucidate the possible function of the mutated amino-acid residues in PAL we built a homology model of PAL based on structural data of HAL and mutagenesis experiments with PAL. The homology model of PAL showed that the active site of PAL resembles the active site of HAL. This allowed us to propose possible roles for the corresponding residues in PAL catalysis.
Chapter
Inhibitors of enzymic and metabolic processes are invaluable tools in biochemical and physiological research, and their application as drugs or pesticides ranges from medicine to agriculture. The information one can extract from their judicious use depends, on the one hand, on the complexity of the system to which they are applied and, on the other hand, on their selectivity for a given target, as well as on their access to this target. Accessibility in this context is meant to include the arrival of the inhibitor at its target site in a state in which it is capable of exerting its inhibitory action. It is obvious that the chances for selectivity of a given inhibitor decrease with the increasing complexity of a system as measured, for example, by the number of enzymic reactions involved and the degree of their interaction and interdependence in the metabolic network of a cell. To illustrate this point, α-aminooxy acetic acid (AOA) is a fairly potent inhibitor of the biosynthesis of phenylpropanoid compounds and has been used in complementation experiments to study the biosynthesis of cyanidin in buckwheat.1
Article
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.
Article
Equations have been developed which provide the basis for the elucidation of the inhibition mechanism of a tight-binding inhibitor by studying the manner in which the substrate interferes with the bincling of the inhibitor to the enzyme. The procedure involves the determination of the pseudo-firstorder rate constants at various concentrations of the substrate and the inhibitor. This method is applicable to very tightly binding inhibitors, including irreversible inhibitors. Methods for the graphical as well as statistical analyses of the data are presented. By the application of these methods, it is demonstrated that coformycin competes with adenosine for adenosine deaminase from calf intestinal mucosa. The kinetic parameters ( ± S. E.) for the bincling of coformycin with adenosine deaminase were determined at 22° and pH 7.4; the second-order rate constant, 1.01 ( ± 0.06) × 1060MP−1 sec−1; the first-order rate constant, 2.2 ( ± 1.0) × 10−4sec−1; and the dissociation constant of the EI complex, 2.2 ( ± 0.76) × 10−10 M.
Article
Both enantiomers of α-aminooxy-β-phenylpropionic acid (AOPP), potent inhibitors of L-phenylalanine ammonia-lyase, and their N-benzyloxycarbonyl (N-BOC) derivatives inhibit anthocyanin formation in developing flowers of Ipomoea tricolor Cav. and Catharanthus roseus Don. as well as in seedlings of Brassica oleracea var. caulo-rapa DC (kohlrabi) and B. oleracea var. capitata L. (red cabbage) with little interference with their normal development. Kohlrabi seedlings tolerate up to 0.3 mM L-AOPP and N-BOC-L-AOPP without a reduction of fresh weight or chlorophyll content, while anthocyanin is reduced to less than 20%.
Article
The hydroxylamine analogue of phenylalanine — α-aminooxy-β-phenyl-propionic acid — is a potent inhibitor of phenylalanine ammonia-lyases (PAL) of both higher plant and fungal origin. The inhibition is competitive, and Ki values were found to be 1.4 nM for the enzyme from buckwheat and 55 nM for the enzyme from the yeast Rhodotorula glutinis. Evidence is presented that the analogue inhibits phenylalanine ammonia-lyase in vivo in buckwheat hypocotyls.
Article
Inhibitors of enzyme-catalysed reactions can be divided into four classes according to the rate and strength of their interactions with enzymes.
Article
Histidine ammonia-lyase (EC 4.3.1.3) catalyzes the nonoxidative elimination of the alpha-amino group of histidine and is closely related to the important plant enzyme phenylalanine ammonia-lyase. The crystal structure of histidase from Pseudomonas putida was determined at 2.1 A resolution revealing a homotetramer with D2 symmetry, the molecular center of which is formed by 20 nearly parallel alpha-helices. The chain fold, but not the sequence, resembles those of fumarase C and related proteins. The structure shows that the reactive electrophile is a 4-methylidene-imidazole-5-one, which is formed autocatalytically by cyclization and dehydration of residues 142-144 with the sequence Ala-Ser-Gly. With respect to the first dehydration step, this modification resembles the chromophore of the green fluorescent protein. The active center is clearly established by the modification and by mutations. The observed geometry allowed us to model the bound substrate at a high confidence level. A reaction mechanism is proposed.
Article
HAL and PAL are closely related enzymes: They both contain a recently discovered highly electrophilic prosthetic group (shown in the picture) at the active site. This observation adds weight to the previously published hypothesis that the reactions catalyzed by HAL and PAL (histidine and phenylalanine ammonia-lyases) have analogous mechanisms.
Article
2-Aminoindan-2-phosphonic acid (3) was synthesized for the first time both directly from 2-indanone (7) as well as by alkylation of ethyl diethoxyphosphorylacetate with 1,2-bis(bromomethyl)benzene (9) via 10–12, respectively. 2-Hydroxyindan-2-phosphonic acid (5) was obtained also from 7 via its diisopropyl ester 8. Compound 3 was found to be a potent inhibitor of the plant enzyme phenylalanine ammonia-lyase (EC 4.3.1.5) both in vitro and in vivo. Of the related 2-aminoindan-2-carboxylic acid (4), compound 5, and 1-aminocyclopentylphosphonic acid (6), only 4 inhibited the enzyme but was far less inhibitory than 3.
Article
High activity of tyrosine aminotransferase (TAT; EC 2.6.1.5) was found in extracts of rosmarinic acid-producing cell cultures of Anchusa officinalis and Coleus blumei, while low activity of tyrosine oxidase was observed. During the culture cycle, TAT activity, phenylalanine ammonia-lyase (PAL) activity and the rate of rosmarinic acid synthesis changed in a coordinated manner. TAT from A. officinalis cultures displayed high substrate specificity for tyrosine but relatively broad specificity toward potential amino acceptors, with equal effectiveness for α-ketoglutarate and oxaloacetate. The enzyme activity was competitively inhibited by l-α-aminooxy-β-phenylpropionic acid (AOPP) with a Ki of 0.8 × 10−6 M. The block of the entry of tyrosine into the tyrosine-derived pathway by AOPP inhibition in vivo confirmed the role of TAT as the entrypoint enzyme of the pathway.
Article
Two analogues of phenylalanine, (±)-2-aminomethyl-3-phenylpropanoic acid and (E)-2-aminomethyl-3-phenylpropenoic acid, were synthesized and found to inhibit buckwheat phenylalanine ammonia-lyase (PAL) competitively with Ki values of 16.5 and 1.3 μM, respectively. They interfered, however, only weakly with light-induced anthocyanin synthesis in etiolated buckwheat seedlings, indicating negligible inhibition of PAL in vivo. In addition, (±)-2-aminomethyl-3-phenylpropanoic acid was a mixed type inhibitor of phenylalanine transamination (Ki=0.93 mM) catalysed by an aminotransferase preparation from mungbean shoots. The two phenylalanine analogues inhibited neither rat liver phenylalanine hydroxylase nor soybean phenylalanyl tRNA synthetase.
Article
Various aspects of the kinetic behavior of the interaction between tight-binding ligands and macromolecules have been examined. The relationships between 50 per cent inhibitory concentration and dissociation constants of the complex are shown for various mechanisms of inhibition, and several methods for the estimation of very low Ki, values are presented. The properties of the Ackermann-Potter plot are examined, and precautions are offered for the usage of the term stoichiometric or titrating inhibition. Both association and dissociation processes between a tight binder and a macromolecule are slow under ordinary laboratory conditions, and steady state rate equations are not applicable. In the presence of a tight-binding inhibitor, the initial velocity of an enzymic reaction depends on the order of addition of the components, and the extent of lag period can be used for the calculation of the rate constant for the slowest step and for diagnosis of the inhibition mechanism. It is also pointed out that the Lineweaver-Burk analysis of competition between two ligands may lead to erroneous conclusions unless the slope of the plot is carefully examined.
Introduction Characteristics of Progress Curves for Slow-Binding Inhibitors Specific Cases of Slow-Binding Inhibition Concluding REmarks
Article
L(-)-, and D(+)-enantiomers of 1-amino-2-phenylethylphosphonic acid (PheP), a phosphonic analogue of phenylalanine, inhibit the activity of L-phenylalanine ammonia-lyase (EC 4.3.1.5) of potato tuber tissue in vitro. The apparent type of inhibition depends on concentration of PheP; as the concentration of D-PheP is raised from 10(-5) M to 2.5 X 10(-3) M, the type of inhibition shifts from competitive through mixed and non-competitive to uncompetitive. L-PheP exerts either a competitive or mixed-type inhibition at low (10(-6)-10(-5) M) or moderate (5 X 10(-5)-2 X 10(-4) M) concentration. Ki for the concentration range of competitive inhibition were 6.5 X 10(-6) M, 5.3 X 10(-5)M and 1.6 X 10(-5) M for L-, D-, and D,L-PheP, respectively. These Ki values are valid for a relatively narrow range of L-Phe concentration (0.2-4 mM) as L-phenylalanine ammonia-lyase does not follow the Michaelis-Menten kinetics of the reaction.
Article
The binding of substrate and product analogs to phenylalanine ammonia-lyase (EC 4.3.1.5) from maize has been studied by a protection method. The ligand dissociation constants, KL, were estimated from the variation with [L] of the pseudo-first-order rate constants for enzyme inactivation by nitromethane. The phenylalanine analogs d- and l-2-aminooxy-3-phenylpropionic acid showed KL, values over 20,000-fold lower than the Km for l-phenylalanine. From these and other KL values it is deduced that when the enzyme binds l-phenylalanine the structural free energy stored in the protein is higher than when it binds the superinhibitors. Models for binding d- and l-phenylalanine and the superinhibitors are described. The enantiomeric pairs are considered to have similar KL values because they pack into the active site in a mirror-image relationship. If the elimination reaction approximates to the least-motion course deduced on stereoelectronic grounds, the mirror-image packing of the superinhibitors into the active site mimics the conformation inferred for a transition state in the elimination. It appears, therefore, that structural changes take place in the enzyme as the transition state conformation is approached causing stored free energy to be released. This lowers the activation free energy for the elimination reaction and accounts for the strong binding by the above analogs.
Article
Phenylalanine ammonia-lyase (EC 4.3.1.5) from parsley is posttranslationally modified by dehydrating its Ser-202 to the catalytically essential dehydroalanine prosthetic group. The codon of Ser-202 was changed to those of alanine and threonine by site-directed mutagenesis. These mutants and the recombinant wild-type enzyme, after treatment with sodium borohydride, were virtually inactive with L-phenylalanine as substrate but catalyzed the deamination of L-4-nitrophenylalanine, which is also a substrate for the wild-type enzyme. Although the mutants reacted about 20 times slower with L-4-nitrophenylalanine than the wild-type enzyme, their Vmax for L-4-nitrophenylalanine was two orders of magnitude higher than for L-phenylalanine. In contrast to L-tyrosine, which was a poor substrate, DL-3-hydroxyphenylalanine (DL-m-tyrosine) was converted by phenylalanine ammonia-lyase at a rate comparable to that of L-phenylalanine. These results suggest a mechanism in which the crucial step is an electrophilic attack of the prosthetic group at position 2 or 6 of the phenyl group. In the resulting carbenium ion, the beta-HSi atom is activated in a similar way as it is in the nitro analogue. Subsequent elimination of ammonia, concomitant with restoration of both the aromatic ring and the prosthetic group, completes the catalytic cycle.
Article
Near-full-length cDNAs for the four phenylalanine ammonia-lyase (PAL) isoenzymes in parsley (Petroselium crispum Nym.) were cloned and the complete amino acid sequences deduced. Fusion proteins with glutathione S-transferase were expressed in Escherichia coli, purified and cleaved. All of the resulting phenylalanine ammonia-lyase proteins, as well as the fusion proteins, were catalytically active. The turnover number of one selected isoenzyme, PAL-1, was estimated to be around 22 s-1 for each active site. In contrast to a certain degree of differential expression in various parts of parsley plants, the four phenylalanine ammonia-lyase isoenzymes exhibited very similar apparent Km values for L-phenylalanine (15-24.5 microM) as well as identical temperature (58 degrees C) and pH (8.5) optima. All of them were competitively inhibited by (E)-cinnamate with similar efficiency (Ki values: 9.1-21.5 microM), lacked cooperative behaviour, and accepted L-tyrosine as a substrate with low affinity (Km values: 2.6-7.8 mM). These results suggest that the occurrence of multiple gene copies has a function other than encoding isoenzymes with different enzyme kinetic properties.
Article
Dextrorotatory 1-amino-3',4'-dichlorobenzylphosphonic acid was found to be a potent inhibitor of the plant enzyme phenylalanine ammonia-lyase both in vitro and in vivo from among the ring-substituted 1-aminobenzylphosphonic acids and other analogues of phenylglycine. A structure activity relationship analysis of the results obtained permits predictions on the geometry of the pocket of the enzyme and is a basis in the strategy of better inhibitor synthesis. (C) 2002 Published by Elsevier Science Ltd.
Specific inhibitors as probes into the biosynthesis and metabolism of aromatic amino acids The Shikimic Acid Pathway
  • N Amrhein
Amrhein, N., 1986. Specific inhibitors as probes into the biosynthesis and metabolism of aromatic amino acids. In: Conn, E.E. (Ed.), The Shikimic Acid Pathway. Rec. Adv. Phytochem., Vol. 20. Plenum Press, New York, London, pp. 83–117.
Tyrosine aminotransferase
  • De-Eknamkul
1-Amino-2-phenylphosphonic acid
  • Janas
Erratum to “Cha, S., 1975. Tight-binding inhibitors-I. Kinetic behavior. Biochem. Pharmacol. 24, 2177–2185”
  • Cha
Inhibitors of phenylalanine ammonia-lyase
  • Zoń