The biosynthesis of shikimate metabolites.
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Amino Acids↗
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Evidence for a reactive gamma-carboxyl group (Glu-418) at the herbicide glyphosate binding site of 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli.
Incubation of 5-enolpyruvylshikimate-3-phosphate synthase, a target for the nonselective herbicide glyphosate (N-(phosphonomethyl)glycine), with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of glycine ethyl ester resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first order kinetics, with a second order rate constant of 2.2 M-1 min-1 at pH 5.5 and 25 degrees C. The inactivation is prevented by preincubation of the enzyme with a combination of the substrate shikimate 3-phosphate plus glyphosate, but not by shikimate 3-phosphate, phosphoenolpyruvate, or glyphosate alone. Increasing the concentration of glyphosate during preincubation resulted in decreasing the rate of inactivation of the enzyme. Complete inactivation of the enzyme required the modification of 4 carboxyl groups per molecule of the enzyme. However, statistical analysis of the residual activity and the extent of modification showed that among the 4 modifiable carboxyl groups, only 1 is critical for activity. Tryptic mapping of the enzyme modified in the absence of shikimate 3-phosphate and glyphosate by reverse phase chromatography resulted in the isolation of a [14C]glycine ethyl ester-containing peptide that was absent in the enzyme modified in the presence of shikimate 3-phosphate and glyphosate. By amino acid sequencing of this labeled peptide, the modified critical carboxyl group was identified as Glu-418. The above results suggest that Glu-418 is the most accessible reactive carboxyl group under these conditions and is located at or close to the glyphosate binding site.
3-Phosphoshikimate 1-Carboxyvinyltransferase↗
Analysis of plant complex matrices by use of nuclear magnetic resonance spectroscopy: St. John's wort extract.
The efficiency of two-dimensional homonuclear (1)H--(1)H correlated spectroscopy and two-dimensional reverse heteronuclear shift correlation spectroscopy (i.e., heteronuclear multiple quantum correlation) in characterizing and evaluating the relative content of herbal extract constituents is demonstrated. These experiments are able to fully assign the proton and carbon resonances of all three classes of constituents present in dried commercial extract of St. John's wort, that is, flavonols, phloroglucinols, and naphthodianthrones, with particular regard to the very unstable phloroglucinols. In addition, shikimic and chlorogenic acids, sucrose, lipids, polyphenols, and traces of solvents of the extractive process (methanol) were also identified. These experiments can be considered to be a very simple and fast analytical method for determining the quality and stability of the titled commercial extract. They represent a generally applicable technique for a rapid screening and a specific measurement of other commercial phytochemicals or, in selected cases, an alternative to the classical analytical techniques such as high-performance thin-layer chromatography, high-performance liquid chromatography, capillary gas chromatography, and electrophoresis.
Drug Stability↗
Arabidopsis seed development and germination is associated with temporally distinct metabolic switches.
While the metabolic networks in developing seeds during the period of reserve accumulation have been extensively characterized, much less is known about those present during seed desiccation and subsequent germination. Here we utilized metabolite profiling, in conjunction with selective mRNA and physiological profiling to characterize Arabidopsis (Arabidopsis thaliana) seeds throughout development and germination. Seed maturation was associated with a significant reduction of most sugars, organic acids, and amino acids, suggesting their efficient incorporation into storage reserves. The transition from reserve accumulation to seed desiccation was associated with a major metabolic switch, resulting in the accumulation of distinct sugars, organic acids, nitrogen-rich amino acids, and shikimate-derived metabolites. In contrast, seed vernalization was associated with a decrease in the content of several of the metabolic intermediates accumulated during seed desiccation, implying that these intermediates might support the metabolic reorganization needed for seed germination. Concomitantly, the levels of other metabolites significantly increased during vernalization and were boosted further during germination sensu stricto, implying their importance for germination and seedling establishment. The metabolic switches during seed maturation and germination were also associated with distinct patterns of expression of genes encoding metabolism-associated gene products, as determined by semiquantitative reverse transcription-polymerase chain reaction and analysis of publicly available microarray data. When taken together our results provide a comprehensive picture of the coordinated changes in primary metabolism that underlie seed development and germination in Arabidopsis. They furthermore imply that the metabolic preparation for germination and efficient seedling establishment initiates already during seed desiccation and continues by additional distinct metabolic switches during vernalization and early germination.
Arabidopsis↗
The occurrence of enzymes involved in phenylpropanoid metabolism in the tapetum fraction of anthers.
The contents of another loculus were separated in a pollen and tapetum fraction. The following enzymes involved in phenylpropanoid metabolism were present in the tapetum fraction: shikimate dehydrogenase; phenylalanine ammonialyase; cinnamic acid 4-hydroxylase; SAM (S-adenosylmethionine): caffeate 3-O-methyltransferase; hydroxycinnamate: CoA ligase; "flavanone synthase"; chalcone-flavanone isomerase; SAM: 3',4'-dihydroxyflavonoid 3'-O-methyltransferase; O-glucosyltransferase. It is postulated, that these enzymes derived from the tapetum catalyze the different steps of phenylpropanoid metabolism at or in cavities of the exine after their transfer into the loculus.
Acyltransferases↗
The characterisation of the shikimate pathway enzyme dehydroquinase from Pisum sativum.
Peptides accounting for 157 residues of the bifunctional shikimate pathway enzyme, dehydroquinase/shikimate dehydrogenase, of Pisum sativum were sequenced. Three of the peptides were homologous to regions in Escherichia coli dehydroquinase and two to E. coli shikimate dehydrogenase. The pea dehydroquinase activity was inhibited by treatment with dehydroquinate plus sodium borohydride, establishing it as a type I dehydroquinase. Synthetic oligonucleotides designed from the amino acid sequence were used as PCR primers to amplify fragments of P. sativum cDNA. DNA sequence analysis showed that these amplified products were derived from dehydroquinase/shikimate dehydrogenase cDNA. The complete amino acid sequence of the dehydroquinase domain has been defined; it is homologous to all other type I dehydroquinases and is N-terminal.
Amino Acid Sequence↗
The shikimate pathway--a metabolic tree with many branches.
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Amino Acids↗
Retrobiosynthetic NMR studies with 13C-labeled glucose. Formation of gallic acid in plants and fungi.
The biosynthesis of gallic acid was studied in cultures of the fungus Phycomyces blakesleeanus and in leaves of the tree Rhus typhina. Fungal cultures were grown with [1-13C]glucose or with a mixture of unlabeled glucose and [U-13C6]glucose. Young leaves of R. typhina were kept in an incubation chamber and were supplied with a solution containing a mixture of unlabeled glucose and [U-13C6]glucose via the leaf stem. Isotope distributions in isolated gallic acid and aromatic amino acids were analyzed by one-dimensional 1H and 13C NMR spectroscopy. A quantitative analysis of the complex isotopomer composition of metabolites was obtained by deconvolution of the 13C13C coupling multiplets using numerical simulation methods. This approach required the accurate analysis of heavy isotope chemical shift effects in a variety of different isotopomers and the analysis of long range 13C13C coupling constants. The resulting isotopomer patterns were interpreted using a retrobiosynthetic approach based on a comparison between the isotopomer patterns of gallic acid and tyrosine. The data show that both in the fungus and in the plant all carbon atoms of gallic acid are biosynthetically equivalent to carbon atoms of shikimate. Notably, the carboxylic group of gallic acid is derived from the carboxylic group of an early intermediate of the shikimate pathway and not from the side chain of phenylalanine or tyrosine. It follows that the committed precursor of gallic acid is an intermediate of the shikimate pathway prior to prephenate or arogenate, most probably 5-dehydroshikimate. A formation of gallic acid via phenylalanine, the lignin precursor, caffeic acid, or 3,4, 5-trihydroxycinnamic acid can be ruled out as major pathways in the fungus and in young leaves of R. typhina. The incorporation of uniformly 13C-labeled glucose followed by quantitative NMR analysis of isotopomer patterns is suggested as a general method for biosynthetic studies. As shown by the plant experiment, this approach is also applicable to systems with low incorporation rates.
Carbon Isotopes↗
The oxidation of D-quinate and related acids by Acetomonas oxydans.
1. Growing cells of a small number of strains of Acetomonas oxydans oxidized d-quinate to 5-dehydroquinate. 2. d-Shikimate was oxidized to 4,5-dihydroxy-3-oxocyclohex-1-ene-1-carboxylate (3-dehydroshikimate, formerly 5-dehydroshikimate). 3. d-Dihydroshikimate was oxidized to the corresponding 5-dehydro compound, but epidihydroshikimate oxidation by growing cells was not observed. 4. Cell-free extracts oxidized d-quinate to 5-dehydroquinate with the consumption of the stoicheiometric amount of oxygen, but oxidation of shikimate and dihydroshikimate did not go to completion. 5. Oxidation of quinate was brought about by a constitutive particulate enzyme probably localized in the cytoplasmic membrane. No evidence was found for the participation of NAD, NADP or free flavine compounds in electron transport, but the system was cytochrome-linked.
Acetobacter↗
Isoflavone, glyphosate, and aminomethylphosphonic acid levels in seeds of glyphosate-treated, glyphosate-resistant soybean.
The estrogenic isoflavones of soybeans and their glycosides are products of the shikimate pathway, the target pathway of glyphosate. This study tested the hypothesis that nonphytotoxic levels of glyphosate and other herbicides known to affect phenolic compound biosynthesis might influence levels of these nutraceutical compounds in glyphosate-resistant soybeans. The effects of glyphosate and other herbicides were determined on estrogenic isoflavones and shikimate in glyphosate-resistant soybeans from identical experiments conducted on different cultivars in Mississippi and Missouri. Four commonly used herbicide treatments were compared to a hand-weeded control. The herbicide treatments were (1) glyphosate at 1260 g/ha at 3 weeks after planting (WAP), followed by glyphosate at 840 g/ha at 6 WAP; (2) sulfentrazone at 168 g/ha plus chlorimuron at 34 g/ha applied preemergence (PRE), followed by glyphosate at 1260 g/ha at 6 WAP; (3) sulfentrazone at 168 g/ha plus chlorimuron at 34 g/ha applied PRE, followed by glyphosate at 1260 g/ha at full bloom; and (4) sulfentrazone at 168 g/ha plus chlorimuron at 34 g/ha applied PRE, followed by acifluorfen at 280 g/ha plus bentazon at 560 g/ha plus clethodim at 140 g/ha at 6 WAP. Soybeans were harvested at maturity, and seeds were analyzed for daidzein, daidzin, genistein, genistin, glycitin, glycitein, shikimate, glyphosate, and the glyphosate degradation product, aminomethylphosphonic acid (AMPA). There were no remarkable effects of any treatment on the contents of any of the biosynthetic compounds in soybean seed from either test site, indicating that early and later season applications of glyphosate have no effects on phytoestrogen levels in glyphosate-resistant soybeans. Glyphosate and AMPA residues were higher in seeds from treatment 3 than from the other two treatments in which glyphosate was used earlier. Intermediate levels were found in treatments 1 and 2. Low levels of glyphosate and AMPA were found in treatment 4 and a hand-weeded control, apparently due to herbicide drift.
Drug Resistance↗
In vitro ruminal fermentation of organic acids common in forage.
Mixed rumen bacteria from cows fed either timothy hay or a 60% concentrate were incubated with 7.5 mM citrate, trans-aconitate, malate, malonate, quinate, and shikimate. Citrate, trans-aconitate, and malate were fermented at faster rates than malonate, quinate, and shikimate. Acetate was the primary fermentation product for all six acids. Quinate and shikimate fermentations gave rist to butyrate, whereas malate and malonate produced significant amounts of propionic acid. High-pressure liquid chromatography of fermentation products from trans-aconitate incubations revealed a compound that was subsequently identified as tricarballylate. As much as 40% of the trans-aconitate acid was converted to tricarballylate, and tricarballylate was fermented slowly. The slow rate of tricarballylate metabolism by mixed rumen bacteria and its potential as a magnesium chelator suggest that tricarballylate formation could be an important factor in the hypomagnesemia that leads to grass tetany.
Animals↗
The phenotype of the Arabidopsis cue1 mutant is not simply caused by a general restriction of the shikimate pathway.
The Arabidopsis thalianachlorophyll a/b binding protein underexpressed (cue1) mutant, which has been isolated in a screen for chlorophyll a/b binding protein (CAB) underexpressors, exhibits a reticulate leaf phenotype combined with delayed chloroplast development and aberrant shape of the palisade parenchyma cells. The affected gene in cue1 is a phosphoenolpyruvate (PEP)/phosphate translocator (PPT) of the plastid inner envelope membrane. The proposed function of the PPT in C3-plants is the import of PEP into the stroma as one of the substrates for the shikimate pathway, from which aromatic amino acids and a variety of secondary plant products derive. The mutant phenotype could be: (i) complemented by constitutive overexpression of a heterologous PPT from cauliflower; and (ii) rescued by overexpression of a C4-type pyruvate,orthophosphate dikinase (PPDK). The latter approach indicates that PEP deficiency within plastids triggers developmental constraints in cue1. The impact of the mutation on aspects of primary and secondary metabolism was assessed in cue1 as well as in the individual transformant lines. The majority of the data obtained in this and an accompanying paper suggest that the mutant phenotype is not simply caused by a general restriction of the shikimate pathway because of a defect in a PPT.
Arabidopsis↗
Aromatic amino acid biosynthesis: gene-enzyme relationships in Bacillus subtilis.
Single step mutants of Bacillus subtilis which required either one or all of the aromatic amino acids for growth were isolated. The relevant gene defect was determined for each mutant by enzyme assays in vitro. A mutant deficient in each enzyme step of aromatic amino acid biosynthesis was found with the exceptions of the shikimate kinase and the phenylalanine and tyrosine transaminases. Representative mutants carrying the defective genes were mapped by deoxyribonucleic acid mediated transformation by reference to the aromatic amino acid gene (aro) cluster and, alternately, to any of the other unlinked aro genes. The genes coding for dehydroquinate synthetase, 3-enol pyruvylshikimate 5-phosphate synthetase, one form of chorismate mutase, and prephenate dehydrogenase are linked to the aro cluster. Except for the previously identified linkage between the genes of 3-deoxy-d-arabino heptulosonic acid 7-phosphate synthetase and one species of chorismate mutase, the other genes involved in this pathway are neither linked to the aro cluster nor to each other.
Amino Acids↗
3-dehydroquinate production by oxidative fermentation and further conversion of 3-dehydroquinate to the intermediates in the shikimate pathway.
3-Dehydroquinate production from quinate by oxidative fermentation with Gluconobacter strains of acetic acid bacteria was analyzed for the first time. In the bacterial membrane, quinate dehydrogenase, a typical quinoprotein containing pyrroloquinoline quinone (PQQ) as the coenzyme, functions as the primary enzyme in quinate oxidation. Quinate was oxidized to 3-dehydroquinate with the final yield of almost 100% in earlier growth phase. Resting cells, dried cells, and immobilized cells or an immobilized membrane fraction of Gluconobacter strains were found to be useful biocatalysts for quinate oxidation. 3-Dehydroquinate was further converted to 3-dehydroshikimate with a reasonable yield by growing cells and also immobilized cells. Strong enzyme activities of 3-dehydroquinate dehydratase and NADP-dependent shikimate dehydrogenase were detected in the soluble fraction of the same organism and partially fractionated from each other. Since the shikimate pathway is remote from glucose in the metabolic pathway, the entrance into the shikimate pathway from quinate to 3-dehydroquinate looks advantageous to produce metabolic intermediates in the shikimate pathway.
Aerobiosis↗
Regulation of enzyme synthesis in the aromatic amino acid pathway of Bacillus subtilus.
The control of the synthesis of certain key enzymes of aromatic amino acid biosynthesis was studied. Tyrosine represses the first enzyme of the 3-deoxy-d-arabino heptulosonic acid 7-phosphate pathway, DAHP synthetase, as well as shikimate kinase and chorismate mutase about fivefold in cultures grown under conditions limiting the synthesis of the aromatic amino acids. A mixture of tyrosine and phenylalanine represses twofold further. Tryptophan does not appear to be involved in the control of these enzymes. The specific activity of at least one early enzyme, dehydroquinase, remains essentially constant under a variety of nutritional supplementations. Two enzymes in the terminal branches are repressed by the amino acids they help to synthesize: prephenate dehydrogenase can be repressed fourfold by tyrosine, and anthranilate synthetase can be repressed over 200-fold by tryptophan. There is no evidence that phenylalanine represses prephenate dehydratase. Regulatory mutants have been isolated in which various enzymes of the pathway are no longer repressible. One class is derepressed for several of the prechorismate enzymes, as well as chorismate mutase and prephenate dehydrogenase. In another mutant, several enzymes of tryptophan biosynthesis are no longer repressible. Thus, the rate of synthesis of enzymes at every stage of the pathway is under control of various aromatic amino acids. Tyrosine and phenylalanine control the synthesis of enzymes involved in the synthesis of the three aromatic amino acids. Each terminal branch is under the control of its end product.
Arabinose↗
New stilbene carboxylic acid from Convolvulus hystrix.
Convolvulus hystrix Vahl. (family Convolvulaceae) is a common wild plant growing in the kingdom of Saudi Arabia. It is used in traditional medicine. Our study resulted in the isolation and structural elucidation of ten natural products from which a stilbene carboxylic acid was isolated for the first time as a new natural product. The isolated products are mainly shikimates in addition to two sterols; beta-sitosterol and stigmasterol, as well as one triterpenoid, oleanolic acid. The shikimate products are vanillin, vanillic acid, syringic acid, ferulic acid, isoferulic acid, isoscopoletin and the new stilbene derivative. The structures were elucidated on the basis of spectral data from 400 MHz 1H NMR, MS, IR and UV. The biogenesis of the new stilbene is discussed.
Carboxylic Acids↗
Evidence for the shikimate-3-phosphate interacting site in the N-terminal domain of 5-enolpyruvyl shikimate-3-phosphate synthase of Bacillus subtilis.
The role of basic amino acid residues that are highly conserved in the N-terminal domain of 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPs) in the binding of the substrate, shikimate-3-phosphate, has been assessed. Lys 19 and Arg 24 in the Bacillus subtilis EPSPs were substituted by glutamic acid and aspartic acid residues respectively by site-directed mutagenesis. Native and the mutant proteins were expressed using a two-vector system and the expressed proteins were purified to near homogeniety. The replacement of either Lys 19 or Arg 24 with a negatively charged residue nearly completely abolished the enzyme activity. The kinetic characterization of the purified wild type and the mutant proteins revealed that the substitution of positively charged residues in the N-terminal domain (K19 and R24) results in reduced affinity for shikimate-3-phosphate (S3P). The results suggest the involvement of these residues in the binding of S3P during enzyme catalysis.
3-Phosphoshikimate 1-Carboxyvinyltransferase↗
The anomalous inhibition of shikimate dehydrogenase by analogues of dehydroshikimate.
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Alcohol Oxidoreductases↗