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PubMed2006

Combined transcript and metabolite profiling of Arabidopsis leaves reveals fundamental effects of the thiol-disulfide status on plant metabolism.

In this study, we used gas chromatography-mass spectrometry analysis in combination with flux analysis and the Affymetrix ATH1 GeneChip to survey the metabolome and transcriptome of Arabidopsis (Arabidopsis thaliana) leaves in response to manipulation of the thiol-disulfide status. Feeding low concentrations of the sulfhydryl reagent dithiothreitol for 1 h at the end of the dark period led to posttranslational redox activation of ADP-glucose pyrophosphorylase and major alterations in leaf carbon partitioning, including an increased flux into major respiratory pathways, starch, cell wall, and amino acid synthesis, and a reduced flux to sucrose. This was accompanied by a decrease in the levels of hexose phosphates, while metabolites in the second half of the tricarboxylic acid cycle and various amino acids increased, indicating a stimulation of anaplerotic fluxes reliant on alpha-ketoglutarate. There was also an increase in shikimate as a precursor of secondary plant products and marked changes in the levels of the minor sugars involved in ascorbate synthesis and cell wall metabolism. Transcript profiling revealed a relatively small number of changes in the levels of transcripts coding for components of redox regulation, transport processes, and cell wall, protein, and amino acid metabolism, while there were no major alterations in transcript levels coding for enzymes involved in central metabolic pathways. These results provide a global picture of the effect of redox and reveal the utility of transcript and metabolite profiling as systemic strategies to uncover the occurrence of redox modulation in vivo.

Arabidopsis↗
PubMed2001

Ubiquinone biosynthesis in microorganisms.

The quinoid nucleus of the benzoquinone, ubiquinone (coenzyme Q; Q), is derived from the shikimate pathway in bacteria and eukaryotic microorganisms. Ubiquinone is not considered a vitamin since mammals synthesize it from the essential amino acid tyrosine. Escherichia coli and other Gram-negative bacteria derive the 4-hydroxybenzoate required for the biosynthesis of Q directly from chorismate. The yeast, Saccharomyces cerevisiae, can either form 4-hydroxybenzoate from chorismate or tyrosine. However, unlike mammals, S. cerevisiae synthesizes tyrosine in vivo by the shikimate pathway. While the reactions of the pathway leading from 4-hydroxybenzoate to Q are the same in both organisms the order in which they occur differs. The 4-hydroxybenzoate undergoes a prenylation, a decarboxylation and three hydroxylations alternating with three methylation reactions, resulting in the formation of Q. The methyl groups for the methylation reactions are derived from S-adenosylmethionine. While the prenyl side chain is formed by the 2-C-methyl-D-erythritol 4-phosphate (non-mevalonate) pathway in E. coli, it is formed by the mevalonate pathway in the yeast.

Bacteria↗
PubMed1999

THE SHIKIMATE PATHWAY.

The shikimate pathway links metabolism of carbohydrates to biosynthesis of aromatic compounds. In a sequence of seven metabolic steps, phosphoenolpyruvate and erythrose 4-phosphate are converted to chorismate, the precursor of the aromatic amino acids and many aromatic secondary metabolites. All pathway intermediates can also be considered branch point compounds that may serve as substrates for other metabolic pathways. The shikimate pathway is found only in microorganisms and plants, never in animals. All enzymes of this pathway have been obtained in pure form from prokaryotic and eukaryotic sources and their respective DNAs have been characterized from several organisms. The cDNAs of higher plants encode proteins with amino terminal signal sequences for plastid import, suggesting that plastids are the exclusive locale for chorismate biosynthesis. In microorganisms, the shikimate pathway is regulated by feedback inhibition and by repression of the first enzyme. In higher plants, no physiological feedback inhibitor has been identified, suggesting that pathway regulation may occur exclusively at the genetic level. This difference between microorganisms and plants is reflected in the unusually large variation in the primary structures of the respective first enzymes. Several of the pathway enzymes occur in isoenzymic forms whose expression varies with changing environmental conditions and, within the plant, from organ to organ. The penultimate enzyme of the pathway is the sole target for the herbicide glyphosate. Glyphosate-tolerant transgenic plants are at the core of novel weed control systems for several crop plants.

Journal Article↗
PubMed1998

On the mechanism of 5-enolpyruvylshikimate-3-phosphate synthase.

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP, a precursor for the aromatic amino acids. This paper examines a recent claim [Studelska, D. R., McDowell, L. M., Espe, M. P., Klug, C. A., and Schaefer, J. (1997) Biochemistry 36, 15555-15560] that the mechanism of EPSP synthase involves two covalent enzyme-intermediates, in complete contrast to a large body of literature that has already proven the involvement of a single noncovalent intermediate. The evidence in the paper of Studelska et al. is examined closely, and unequivocal proof is provided that those authors' NMR assignments to covalent structures are in error, and that in fact the species they observed were simply the product EPSP and a side-product EPSP ketal. Since those authors used rotational-echo double-resonance (REDOR) solid-state NMR to measure intermolecular and intramolecular distances in the proposed covalent intermediates, we have used REDOR to measure the same distances in enzyme-free and enzyme-bound preparations of purified EPSP, and enzyme-free preparations of purified EPSP ketal. The distance between the shikimate ring phosphorus atom and C8 in enzyme-free EPSP is 6.6 +/- 0.1 A, which lengthens to 7.4 +/- 0.1 A in the presence of the enzyme, and in enzyme-free EPSP ketal is 5.6 +/- 0.1 A. These are entirely consistent with those measured by Studelska et al., which were 7.5 +/- 0.5 A for a putative enzyme-enolpyruvyl species and 6.1 +/- 0.3 A for a putative enzyme-ketal species.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2006

Proteomic examination of Ralstonia eutropha in cellular responses to formic acid.

In this study, Ralstonia eutropha was used to elucidate protein changes in response to formic acid. Sixty-three differentially expressed proteins in relation to formic acid in R. eutropha were found with 1-D PAGE and nano-LC-MS/MS. Among the proteins with decreased expression, four were involved in the shikimate pathway and three proteins in the pyrimidine biosynthesis pathway. With the increased expression of proteins, a dramatic change occurred in the induction of ion transporters in relation to maintenance of the acid-base balance. A detoxification process of formic acid in the bacteria might be related to a membrane enzyme, formate hydrogenylase. Three proteins in polyhydroxyalkanoate synthesis were enhanced and five proteins in glutathione biosynthesis increased in response to formic acid. Three enzymes in mevalonate biosynthesis and heat shock proteins were also elevated in the cells. Therefore, formic acid might have an inhibitory effect on aromatic amino acid production and pyrimidine biosynthesis in R. eutropha. R. eutropha cells seemed to attempt to overcome the effects of formic acid by increasing ion transporters and proteins that metabolized formic acid.

Bacterial Proteins↗
PubMed2000

Isozyme diversity in cassava cultivars (Manihot esculenta Crantz).

Isoenzyme electrophoresis was used as a method to determine genetic diversity in various M. esculenta cultivars collected in the Southwestern (SW) and Northwestern (NW) regions of the State of Parana, in the South region of Brazil, and in cultivars produced at the Agronomic Institute of Campinas (IAC), São Paulo State, Southeastern region of Brazil. The cultivars have been maintained by vegetative propagation for 5 years and are useful in production programs. A total of 28 loci in the acid phosphatase (ACP; EC 3.1.3.2), esterases (EST; EC 3.1.1.1), malate dehydrogenase (MDH; EC 1.1.1.37), and shikimate dehydrogenase (SKDH; EC 1.1.1.15) isozymes was analyzed. The proportion of polymorphic loci for NW, SW, and IAC cultivars was 57.14, 50.0, and 53.6% respectively. Genetic diversity calculated by Nei's genetic identity (I) showed high I values for the three M. esculenta subpopulations. The high degree of polymorphism expressed by cassava cultivars is highly relevant to stimulate breeding programs with M. esculenta species.

Alleles↗
PubMed2001

A metabolic node in action: chorismate-utilizing enzymes in microorganisms.

The shikimate pathway has been described as a metabolic tree with many branches that led to the synthesis of an extensive range of products. This pathway is present only in bacteria, fungi, and plants. While there is only little difference in the sequence of the chemical reactions of the pathway, significant differences exist in terms of organization and regulation. In the main trunk of the shikimate pathway, D-erythrose 4-phosphate and phosphoenolpyruvate are converted via shikimate to chorismate. Chorismate is the common precursor for the biosynthesis of the aromatic amino acids, phenylalanine, tyrosine, and tryptophan, but also for other products as diverse as folate cofactors, benzoid and naphthoid coenzymes, phenazines, and siderophores. Five chorismate-utilizing enzymes have been characterized in microorganisms: chorismate mutase, anthranilate synthase, aminodeoxychorismate synthase, isochorismate synthase, and chorismate pyruvate-lyase. In this review these enzymes are discussed in terms of the corresponding gene structures and regulation, nucleotide and protein sequences, protein structures, and reaction mechanisms. The main emphasis is on transcriptional and posttranslational regulatory mechanisms, in view of how a microbial cell exploits its chorismate pool in diverse anabolic pathways. Comparison of the chorismate-utilizing enzymes has shown that some of them share sequence similarity, suggesting divergent evolution and commonality in reaction mechanisms. However, other chorismate-utilizing enzymes are examples of convergent evolution toward similar reaction capabilities.

Anthranilate Synthase↗
PubMed1999

Cloning and characterization of an esophageal-gland-specific chorismate mutase from the phytoparasitic nematode Meloidogyne javanica.

Root-knot nematodes are obligate plant parasites that alter plant cell growth and development by inducing the formation of giant feeder cells. It is thought that nematodes inject secretions from their esophageal glands into plant cells while feeding, and that these secretions cause giant cell formation. To elucidate the mechanisms underlying the formation of giant cells, a strategy was developed to clone esophageal gland genes from the root-knot nematode Meloidogyne javanica. One clone, shown to be expressed in the nematode's esophageal gland, codes for a potentially secreted chorismate mutase (CM). CM is a key branch-point regulatory enzyme in the shikimate pathway and converts chorismate to prephenate, a precursor of phenylalanine and tyrosine. The shikimate pathway is not found in animals, but in plants, where it produces aromatic amino acids and derivative compounds that play critical roles in growth and defense. Therefore, we hypothesize that this CM is involved in allowing nematodes to parasitize plants.

Amino Acid Sequence↗
PubMed1995

New EPSP synthase inhibitors: synthesis and evaluation of an aromatic tetrahedral intermediate mimic containing a 3-malonate ether as a 3-phosphate surrogate.

A new analog of the EPSP synthase enzyme reaction intermediate 1, containing a 3-malonate ether moiety in place of the usual 3-phosphate group, was synthesized from 3,5-dihydroxybenzoic acid. This simple, synthetically accessible aromatic compound (5) is an effective competitive inhibitor versus S3P with an apparent Ki of 1.3 +/- 0.22 microM. This result demonstrates that a simple benzene ring can be a suitable achiral substitute for the more complex shikimate ring in the design of EPSP synthase inhibitors. Furthermore, the greater potency of 5 versus the phenol 6, glycolate 7 and the gallic acid analog 8 demonstrates the requirement for multiple anionic charges at the dihydroxybenzoate 5-position in order to attain effective inhibition of this enzyme. However, this 3-malonate ether substituted compound was at least 10-fold less effective as a bisubstrate inhibitor than the corresponding 3-phosphate. This suggests that tetrahedral intermediate mimics possessing a 3-malonate ether moiety are less effective than their corresponding 3-phosphates in accessing the optimal enzyme conformation stabilizing 1.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1984

5-Enolpyruvylshikimate-3-phosphate synthase of Klebsiella pneumoniae 2. Inhibition by glyphosate [N-(phosphonomethyl)glycine].

The broad-spectrum, non-selective herbicide glyphosate [N-(phosphonomethyl)glycine] is a potent inhibitor of highly purified 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase, EC 2.5.1.19) of Klebsiella pneumoniae. The inhibition is competitive with phosphoenolpyruvate (PEP) with Ki = 1 microM at pH 6.8 and non-competitive with shikimate 3-phosphate, EPSP, and inorganic phosphate. Non-herbicidal analogues of glyphosate, such as aminomethylphosphonic acid, bis-N-(phosphonomethyl)glycine and iminodiacetic acid, do not inhibit the enzyme. Inhibition of EPSP synthase by glyphosate strongly increases with increasing pH. Glyphosate protects the enzyme against inactivation by phenylglyoxal, 3-bromopyruvate, and N-ethylmaleimide. It is proposed that glyphosate binds to the PEP-binding site of EPSP synthase as a transition-state analogue of PEP. Other PEP-utilizing enzymes were not found to be subject to inhibition by glyphosate.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2005

Characterization of the altered anthranilate synthase in 5-methyltryptophan-resistant rice mutants.

In an earlier investigation, homologous mutant lines resistant to growth inhibition by 5-methyltryptophan (5MT) were selected from a callus that had been irradiated with a 50-Gy gamma ray during embryo culture. In order to identify the 5MT-resistant mechanism, we have continued our investigations of these mutant lines and studied the anthranilate synthase activity of the M5) advanced lines by direct fluorometric detection of the anthranilate formed in both control plants and mutant lines grown on 500 microM 5MT. The anthranilate synthase activity of the mutant plants was 2.2- to 3-fold higher than that of the control. In a kinetic analysis with tryptophan, an anthranilate synthase of the mutant lines was insensitive to feedback inhibition. These lines showed an enhanced accumulation of storage proteins and amino acids. The increased rates of protein synthesis in the mutant lines, relative to that of the control seeds, were 17-28.5%. The amino acid contents were 2.4-fold (MRI-40-2) to 2.6-fold (MRI-110-6) higher in the MRI lines than in the control seeds, and 2.4-fold (MRII-12-5) to 3.5-fold (MRII-8-1) higher in the MRII lines than in the control seeds. Significant increases among the amino acids of the MR lines were observed for tryptophan, phenylalanine, and tyrosine, which had been biosynthesized through the shikimate pathway. The transcript levels of putative OASA2, which is one of the key-regulating enzyme subunits in the tryptophan biosynthesis pathway, were studied in the control and 5MT-resistant mutant lines subjected to inhibition by two tryptophan analogs (5MT and alphaMT) and to other abiotic stresses (ABA, NaCl, and cold). The putative OASA2 gene in the 5MT-resistant mutant lines was highly expressed in at a low 5MT concentration and at an early stage of the 5MT and alphaMT treatments. However, mRNA accumulation of the putative OASA2 gene in the mutant plants gradually decreased when the plants were subjected to abiotic stresses such as NaCl and cold. These results indicated that the 5MT resistance in the mutant lines is due to altered anthranilate synthase forms.

Amino Acids↗
PubMed2026

Integrated ¹H-NMR Metabolomics and Growth Kinetics Uncover Three Distinct Metabolic Scenarios in Lactiplantibacillus pentosus P7 Fermentation of Plant-Derived Prebiotics.

Lactic acid bacteria (LAB) drive a broad range of food and biotechnological fermentations, the outcomes of which depend not only on the bacterial genotype but also on the chemical composition of the fermentation substrate. To resolve how a single strain reorganises chemically distinct plant matrices, we profiled fermentations of Lactiplantibacillus pentosus P7 (GenBank JBLMKZ000000000) on garlic, onion, and kiwifruit extracts prepared in water and 70% ethanol, using growth kinetics combined with solvent-suppressed 500-MHz proton nuclear magnetic resonance metabolomics over 48 h, and integrated the data with whole-genome pathway annotations. Three substrate-specific metabolic scenarios emerged. On garlic, P7 grew vigorously, with the water extract exceeding the de Man-Rogosa-Sharpe reference medium at every time point (peak ΔOD₆₀₀ of 9.38 versus 8.52 at 24 h) and accumulating sorbose, rhamnose, and the aromatic amino acids phenylalanine and tryptophan (3.04- to 3.70-fold increases), providing first metabolic evidence consistent with the strain's four-copy aroE shikimate-dehydrogenase expansion. On onion, the lowest cell density coincided with the highest lactate output of the dataset (5.21-fold rise at 48 h), transient 5-hydroxymethylfurfural reduction, and accumulation of acetoin and 1,3-propanediol, mapping onto a redundant set of pyridine-nucleotide-dependent oxidoreductases and a pdu-independent diol pathway. On kiwifruit, citrate accumulated 8.9-fold at 16 h and then declined, consistent with an intact citCDEFG citrate-lyase operon paired with absence of canonical oxidative tricarboxylic acid enzymes. The optimal extraction solvent was substrate-dependent, water for garlic and ethanol for onion and kiwifruit. Overall, these results show that substrate chemistry, rather than strain identity, dictates which genome-encoded pathways P7 engages, establishing P7 as a versatile, substrate-tunable platform for the functional fermentation and biorefining of furanic-rich substrate streams. Raw NMR data and ISA-Tab metadata are available via MetaboLights with identifier MTBLS14463.

Lactiplantibacillus pentosus↗
PubMed1990

Mechanism of inactivation of Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase by o-phthalaldehyde.

In order to identify the essential reactive amino acid residues of 5-enolpyruvoylshikimate-3-phosphate synthase, a target for the nonselective herbicide glyphosphate (N-phosphonomethylglycine), chemical modification studies with o-phthalaldehyde were undertaken. Incubation of the enzyme with the reagent resulted in a time-dependent loss of enzyme activity. The inactivation followed first-order and saturation kinetics with a Kinact of 25 microM and a maximum rate constant of 0.34 min-1. The inactivation was prevented by preincubation of the enzyme with the substrates shikimate 3-phosphate, 5-enolpyruvoylshikimate 3-phosphate, or by a combination of shikimate 3-phosphate plus glyphosate, but not by phosphoenolpyruvate or glyphosate alone. Absorbance and fluorescence spectra studies indicate that complete inactivation of the enzyme resulted from the formation of two isoindole derivatives per molecule of enzyme. Tryptic mapping of the enzyme modified in the absence of shikimate 3-phosphate and glyphosate resulted in the isolation of two peptides which were not found for the enzyme modified in the presence of shikimate 3-phosphate and glyphosate. Analyses of these two peptides indicate that Lys-22 and Lys-340 were the modified sites. The amino acid sequences around these residues are conserved in bacterial, fungal, as well as plant enzymes, suggesting that these regions may constitute part of the enzyme active site.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1985

Phytoalexins.

Plants respond to infection by accumulating low-molecular-weight antimicrobial stress metabolites called phytoalexins. The phytoalexins are generally lipophilic substances that are products of a plant's secondary metabolism, and they often accumulate at infection sites to concentrations which are inhibitory to the development of fungi and bacteria. Resistance and susceptibility in plants are not determined by the presence or absence of genetic information for resistance mechanisms, including biosynthetic pathways for phytoalexin synthesis, but, rather, by the speed with which the information is expressed, the activity of the gene products, and the magnitude of the resistance response. Unlike the antibody-antigen component of the immune system in animals, low specificity is the general rule for the induction of phytoalexin accumulation and their activity against microorganisms. Annual plants can be systemically immunized against diseases caused by fungi, bacteria, and viruses by restricted infection with the pathogens, avirulent forms of pathogens, or compounds formed in immunized plants. Immunization induces plants to respond rapidly to infection with a multicomponent resistant response. The biosynthesis and accumulation of phytoalexins is one component of this resistant response. Resistance may be elicited by components in the walls and cell surfaces of fungi and bacteria and by compounds liberated from cells, their walls, or surfaces. Resistance can be enhanced or suppressed by products produced by the pathogen, the host, or by their interaction. The successful pathogen avoids recognition by the plant as nonself, suppresses the resistance response, or detoxifies its products. The actors in this play for survival on the metabolic level include the shikimate, acetate-malonate, and acetate-mevalonate pathways; glucans; oligogalacturonates; glycoproteins; lipopolysaccharides; and poly-unsaturated fatty acids. The play is directed by the genetic information of host and pathogen, and this direction is at the level of recognition and not by the presence or absence of mechanisms to contain the development of infectious agents.

Bacteria↗
PubMed2005

CO(2)-induced total phenolics in suspension cultures of Panax ginseng C. A. Mayer roots: role of antioxidants and enzymes.

The effects of different concentrations of CO(2) (1%, 2.5% and 5%) on the antioxidant capacity, total phenols, flavonoids, protein content and phenol biosynthetic enzymes in roots of Panax ginseng were studied in bioreactor (working volume 4 l) after 15, 30 and 45 days. CO(2) induced accumulation of total phenolics in a concentration and duration dependent manner. Total phenols, flavonoids and 1,1-diphenyl-2-picrylhydrazyl (DPPH) activity increased 60%, 30% and 20% at 2.5% CO(2) after 45 days compared to control in P. ginseng roots which indicated that phenolics compounds played an important role in protecting the plants from CO(2). Hypothesizing that increasing the phenolic compounds in roots of P. ginseng may increase its nutritional functionality; we investigated whether pentose phosphate pathway (PPP), shikimate/phenylpropanoid pathway enzymes have a role in phenolics mobilization in P. ginseng roots. Fresh weight (FW), dry weight (DW) and growth ratio was increased at 1% and 2.5% CO(2) only after 45 days, however, unaffected after 15 and 30 days. Results also indicated that high CO(2) progressively stimulated the activities of glucose 6 phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49), shikimate dehydrogenase (SKDH, E.C. 1.1.1.25), phenylalanine ammonia lyase (PAL, E.C. 4.3.1.5), cinnamyl alcohol dehydrogenase (CAD, E.C. 1.1.1.195), caffeic acid (CA) peroxidase and chlorogenic acid (CGA) peroxidase after 15, 30 and 45 days. Increased CO(2) levels resulted in increases in accumulation of total protein (45%), non-protein thiol (NP-SH) (30%) and cysteine contents (52%) after 45 days compared to control and increased activities of beta-glucosidase (GS, E.C. 3.2.1.21) and polyphenol oxidase (PPO, E.C. 1.10.3.2) in P. ginseng roots indicated that they played an important role in protecting the plants from CO(2). These results strongly suggest that high concentration of CO(2) delivered to ginseng root suspension cultures induced the accumulation of total phenolics possessing high antioxidant properties probably useful for human health. Therefore, roots of P. ginseng are considered as a good source of phenolics compounds with high antioxidants capacity and can be produced on a large scale.

Alcohol Oxidoreductases↗
PubMed2005

Initial evaluation of sugarcane as a production platform for p-hydroxybenzoic acid.

Sugarcane (Saccharum hybrids) was evaluated as a production platform for p-hydroxybenzoic acid using two different bacterial proteins (a chloroplast-targeted version of Escherichia coli chorismate pyruvate-lyase and 4-hydroxycinnamoyl-CoA hydratase/lyase from Pseudomonas fluorescens) that both provide a one-enzyme pathway from a naturally occurring plant intermediate. The substrates for these enzymes are chorismate (a shikimate pathway intermediate that is synthesized in plastids) and 4-hydroxycinnamoyl-CoA (a cytosolic phenylpropanoid intermediate). Although both proteins have previously been shown to elevate p-hydroxybenzoic acid levels in plants, they have never been evaluated concurrently in the same laboratory. Nor are there any reports on their efficacy in stem tissue. After surveying two large populations of transgenic plants, it was concluded that the hydratase/lyase is the superior catalyst for leaf and stem tissue, and further studies focused on this pathway. p-Hydroxybenzoic acid was quantitatively converted to glucose conjugates by endogenous uridine diphosphate (UDP)-glucosyltransferases and presumably stored in the vacuole. The largest amounts detected in leaf and stem tissue were 7.3% and 1.5% dry weight (DW), respectively, yet there were no discernible phenotypic abnormalities. However, as a result of diverting carbon away from the phenylpropanoid pathway, there was a severe reduction in leaf chlorogenic acid, subtle changes in lignin composition, as revealed by phloroglucinol staining, and an apparent compensatory up-regulation of phenylalanine ammonia-lyase. Although product accumulation in the leaves at the highest level of gene expression obtained in the present study was clearly substrate-limited, additional experiments are necessary before this conclusion can be extended to the stalk.

Journal Article↗
PubMed1974

Mis-regulation of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase does not account for growth inhibition by phenylalanine in Agmenellum quadruplicatum.

The growth of the blue-green bacterium, Agmenellum quadruplicatum, is inhibited in the presence of l-phenylalanine. This species has a single, constitutively synthesized 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthetase. l-Phenylalanine inhibits DAHP synthetase non-competitively with respect to both substrate reactants. Other aromatic amino acids do not inhibit the activity of DAHP synthetase. A common expectation for branch-point enzymes such as DAHP synthetase is a balanced pattern of feedback control by all of the ultimate end products. It seemed likely that growth inhibition might equate with defective regulation within the branched aromatic pathway. Accordingly, the possibility was examined that mis-regulation of DAHP synthetase by l-phenylalanine in wild-type cells causes starvation for precursors of the other aromatic end products. However, the molecular basis for growth inhibition cannot be attributed to l-phenylalanine inhibition of DAHP synthetase for the following reasons: (i) DAHP synthetase enzymes from l-phenylalanine-resistant mutants are more, rather than less, sensitive to feedback inhibition by l-phenylalanine. (ii) Shikimate not only fails to antagonize inhibition, but is itself inhibitory. (iii) Neither the sensitivity nor the completeness of l-phenylalanine inhibition of the wild-type enzyme in vitro appears sufficient to account for the potent inhibition of growth in vivo by l-phenylalanine. The dominating effect of l-phenylalanine in the control of DAHP synthetase appears to reflect a mechanism that prevents rather than causes growth inhibition by l-phenylalanine. The alteration of the control of DAHP synthetase in mutants selected for resistance to growth inhibition by l-phenylalanine did indicate that the cause for this metabolite vulnerability can be localized within the aromatic amino acid pathway. Apparently, an aromatic intermediate (between shikimate and the end products) accumulates in the presence of l-phenylalanine, causing toxicity by some unknown mechanism. It is concluded that phenylpyruvate, potentially formed by transamination of l-phenylalanine, is an unlikely cause of growth inhibition. Although several significant questions remain unanswered, our results suggest that single-effector control of DAHP synthetase, the first regulatory enzyme activity of a branched pathway, may be more appropriate than it would seem a priori.

Amino Acid Isomerases↗