Enzyme Of Shikimate Pathway Research Articles

Experiences with the shikimate-pathway enzymes as targets for rational drug design.

The background and current context of work on the shikimate-pathway enzymes as potential targets for anti-bacterial, anti-fungal and anti-parasitic drugs is reviewed. Recent work on the third enzyme of the pathway, dehydroquinase, which occurs in two structurally and mechanistically distinct forms, is used to illustrate the present state of studies into rational drug design.

Twists and turns: a tale of two shikimate-pathway enzymes.

We are studying two enzymes from the shikimate pathway, dehydroquinate synthase (DHQS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Both enzymes have been the subject of numerous studies to elucidate their reaction mechanisms. Crystal structures of DHQS and EPSPS in the presence and absence of substrates, cofactors and/or inhibitors are now available. These structures reveal movements of domains, rearrangements of loops and changes in side-chain positions necessary for the formation of a catalytically competent active site. The potential for using complementary small-angle X-ray scattering (SAXS) studies to confirm the presence of these structural differences in solution has also been explored. Comparative analysis of crystal structures, in the presence and absence of ligands, has revealed structural features critical for substrate-binding and catalysis. We have also analysed these structures by generating GRID energy maps to detect favourable binding sites. The combination of X-ray crystallography, SAXS and computational techniques provides an enhanced analysis of structural features important for the function of these complex enzymes.

One-step purification of 5-enolpyruvylshikimate-3-phosphate synthase enzyme from Mycobacterium tuberculosis

Currently, there are 8 million new cases and 2 million deaths annually from tuberculosis, and it is expected that a total of 225 million new cases and 79 million deaths will occur between 1998 and 2030. The reemergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons, and the proliferation of multi-drug-resistant strains have created a need to develop new antimycobacterial agents. The existence of homologues to the shikimate pathway enzymes has been predicted by the determination of the genome sequence of Mycobacterium tuberculosis. We have previously reported the cloning and overexpression of M. tuberculosis aroA-encoded EPSP synthase in both soluble and active forms, without IPTG induction. Here, we describe the purification of M. tuberculosis EPSP synthase (mtEPSPS) expressed in Escherichia coli BL21(DE3) host cells. Purification of mtEPSPS was achieved by a one-step purification protocol using an anion exchange column. The activity of the homogeneous enzyme was measured by a coupled assay using purified shikimate kinase and purine nucleoside phosphorylase proteins. A total of 53 mg of homogeneous enzyme could be obtained from 1 L of LB cell culture, with a specific activity value of approximately 18 U mg −1 . The results presented here provide protein in quantities necessary for structural and kinetic studies, which are currently underway in our laboratory.

Differential expression of 5-enol-pyruvyl-shikimate-3-phosphate synthase isoforms in elicitor-treated, cultured maize cells.

The expression of two 5-enol-pyruvyl-shikimate-3-phosphate synthase (EC 2.5.1.19) isoforms was investigated in Zea mays L. suspension-cultured cells following exposure to a fungal elicitor. Activity levels of isozyme II specifically increased soon after treatment, in strict connection with induction of phenylalanine ammonia-lyase (PAL) and attainment of a new free-phenylalanine homeostasis at a higher concentration. However, a few days later, activity of the other enzyme form was also significantly enhanced, concomitant with a sharp rise in overall amino acid content, a further increase in PAL level and a resumption of cell lysis. Besides strengthening the hypothesis that an entire set of genes encoding for shikimate pathway enzymes (whose expression is specifically involved in plant dynamic defence) may exist, a general change in the levels of several amino acids seems to point towards a reprogramming of their metabolism in elicited cells.

Cloning of genomic DNA of rice 5-enolpyruvylshikimate 3-phosphate synthase gene and chromosomal localization of the gene

The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0 x10(4) clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indicajaponica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.

The molecular architecture of the shikimate pathway enzymes: new insights into mechanisms and drug design

The molecular architecture of the shikimate pathway enzymes: new insights into mechanisms and drug design

A Secondary β Deuterium Kinetic Isotope Effect in the Chorismate Synthase Reaction

Chorismate synthase (EC 4.6.1.4) is the shikimate pathway enzyme that catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. The enzyme reaction is unusual because it involves a trans-1,4 elimination of the C-3 phosphate and the C-6 proR hydrogen and it has an absolute requirement for reduced flavin. Several mechanisms have been proposed to account for the cofactor requirement and stereochemistry of the reaction, including a radical mechanism. This paper describes the synthesis of [4-2H]EPSP and the observation of kinetic isotope effects using this substrate with both Neurospora crassa and Escherichia coli chorismate synthases. The magnitude of the effects were D(V) = 1.08 ± 0.01 for the N. crassa enzyme and 1.10 ± 0.02 on phosphate release under single-turnover conditions for the E. coli enzyme. The effects are best rationalised as substantial secondary β isotope effects. It is most likely that the C(3)-O bond is cleaved first in a nonconcerted E1 or radical reaction mechanism. Although this study alone cannot rule out a concerted E2-type mechanism, the C(3)-O bond would have to be substantially more broken than the proR C(6)-H bond in a transition state of such a mechanism. Importantly, although the E. coli and N. crassa enzymes have different rate limiting steps, their catalytic mechanisms are most likely to be chemically identical.

Reversal of glyphosate inhibition of Sandersonia aurantiaca flower senescence with aromatic amino acids

Glyphosate (N-(phosphonomethyl) glycine) is a broad spectrum post-emergence herbicide. This herbicide inhibits the shikimate pathway enzyme EPSP synthase (5-enol pyruvylshikimate 3-phosphate synthase), thereby interfering with aromatic amino acid metabolism. During preliminary investigations with inhibitors of protein and amino acid biosynthesis, we noticed that vase solutions containing glyphosate altered the normal pattern of Sandersonia aurantiaca flower senescence. Further studies showed that although glyphosate (2 mM) was toxic to all green tissue on the flower stem, the senescence of mature flowers (no green tissue) was delayed. Glyphosate-treated flowers did not fade but stayed a bright orange colour and the compressive strength of the flowers was greater (the flowers were less wilted) than the control flowers that were held in water. Treatment of flowers with vase solutions of phenylalanine (2 mM) and tyrosine (2 mM) in the presence of glyphosate reversed the beneficial effect that glyphosate treatment had on flower senescence. The data indicate that a lack of aromatic amino acids may be the cause of delayed fading and wilting of glyphosate-treated sandersonia flowers.

Evidence for the shikimate pathway in apicomplexan parasites.

Parasites of the phylum Apicomplexa cause substantial morbidity, mortality and economic losses, and new medicines to treat them are needed urgently. The shikimate pathway is an attractive target for herbicides and antimicrobial agents because it is essential in algae, higher plants, bacteria and fungi, but absent from mammals. Here we present biochemical, genetic and chemotherapeutic evidence for the presence of enzymes of the shikimate pathway in apicomplexan parasites. In vitro growth of Toxoplasma gondii, Plasmodium falciparum (malaria) and Cryptosporidium parvum was inhibited by the herbicide glyphosate, a well-characterized inhibitor of the shikimate pathway enzyme 5-enolpyruvyl shikimate 3-phosphate synthase. This effect on T. gondii and P. falciparum was reversed by treatment with p-aminobenzoate, which suggests that the shikimate pathway supplies folate precursors for their growth. Glyphosate in combination with pyrimethamine limited T. gondii infection in mice. Four shikimate pathway enzymes were detected in extracts of T. gondii and glyphosate inhibited 5-enolpyruvyl shikimate 3-phosphate synthase activity. Genes encoding chorismate synthase, the final shikimate pathway enzyme, were cloned from T. gondii and P. falciparum. This discovery of a functional shikimate pathway in apicomplexan parasites provides several targets for the development of new antiparasite agents.

Chemical modification monitored by electrospray mass spectrometry: a rapid and simple method for identifying and studying functional residues in enzymes.

A simple method to identify functional amino acids in enzymes is described. This method is based on the mass spectrometric detection of molecular weight changes as the consequence of chemical modification of enzymes with group-specific reagents. Here we report the use of phenylglyoxal, trinitrobenzene sulfonic acid, tetranitromethane and diethylpyrocarbonate to identify functional amino acid residues. Precise information is obtained about the stoichiometry of reaction, and a relationship between the loss of enzyme activity and the amount of chemical modification is easily established. Modification sites are located by proteolytic digestion of the modified enzyme, followed by peptide mapping based on high-pressure liquid chromatography using an electrospray mass spectrometer as an on-line detector. In comparison with more conventional methods, protein modification is monitored directly without the need to use radioactively or spectrally labelled reagents. The methodology is limited only by the stability of the chemically modified species produced. The method has been used to characterise the active sites of several shikimate pathway enzymes, and the results obtained have been confirmed by site-directed mutagenesis and X-ray crystallography.

Properties of the 5- enol-pyruvyl-shikimate-3-phosphate synthase isoforms isolated from maize cultured cells

Two isoforms of the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate synthase, previously purified from maize cultured cells and both found to be functionally located in the plastid while showing a different pattern of expression during the culture growth cycle, were characterized with respect to physical and functional properties. A high degree of similarity was found as to their structural features, with the only exception of a slight difference in molecular mass. Both enzyme activities were extremely susceptible to the inhibition brought about by the herbicide glyphosate, and not subjected to feed-back regulation by aromatic amino acids or shikimate pathway intermediates. A more pronounced difference was evident in thermal stability, catalytic efficiency as judged from the comparison of catalytic constants, affinities for the two substrates and activation energy values. The isozyme detectable in actively proliferating cells, when the plant cell demand for aromatic amino acids increases, proved to be the more stable and efficient. Data are consistent with the hypothesis of an isoform-based mechanism of enzyme level modulation in plant aromatic metabolism.

Herbicidally Active Derivatives of Aminomethylenebis-Phosphonic Acid-Mode of Action and Structure - Activity Relationship

Abstract: (N-pyridylamino)methylenebisphosphonates exhibit strong herbicidal activity which may be reversed by supplementation of the growth media with aromatic amino acids. They appeare to be the inhibitors of aromatic amino acids biosynthesis acting as inhibitors of DAHP synthase the first enzyme of shikimate pathway. Over 40 analogues of these acids were synthesized in order to determine the structure-activity relationship.

Only the Mature Form of the Plastidic Chorismate Synthase Is Enzymatically Active.

Coding regions of a cDNA for precursor and mature chorismate synthase (CS), a plastidic enzyme, from Corydalis sempervirens were expressed in Escherichia coli as translational fusions to glutathione-S-transferase. Fusion proteins were purified, and precursor and mature forms of CS were then released by proteolytic cleavage with factor Xa. Although mature CS was enzymatically active after release, activity could be detected neither for the precursor CS nor for corresponding glutathione-S-transferase fusion proteins. In contrast, two other shikimate pathway enzymes (shikimate kinase and 5-enol-pyruvylshikimate-3-phosphate synthase) have previously been shown to be as enzymatically active as their respective higher molecular weight precursors. By expression of unfused, mature CS from C. sempervirens in E. coli, it was possible to obtain large quantities of enzymatically active CS protein compared to yields from plant cell cultures. Expression levels in E. coli approached 1% of total soluble protein. No differences were found between authentic CS isolated from cell cultures and CS expressed in and purified from E. coli, which made possible a more detailed biochemical characterization of CS. Quaternary structure analysis of the purified mature CS indicated that the enzyme exists as a dimer, in contrast to the active tetrameric structures determined for E. coli and Neurospora crassa enzymes.

Glyphosate tolerance in maize (Zea mays L.). 2. Selection and characterization of a tolerant somaclone

The progeny of 104 regenerated maize plants were screened for tolerance to the safe broad-spectrum herbicide glyphosate during seed germination and early growth. Seven somaclones showed varying degrees of resistance to the application of the herbicide at 1.2 mM (0.1 kg a.i. in 400 1 ha-1 of water). Plants capable of a normal growth following treatment with 2.4 mM (0.2 kg ha-1) glyphosate at the three leaf stage were selfed, and their progeny analyzed. A family able to tolerate the exposure to glyphosate at 2.4 mM was isolated and shown to maintain a photosynthetic rate comparable with control after the application of the herbicide. The selfed progeny of the tolerant somaclone was characterized as to the properties of two targets of glyphosate, the shikimate pathway enzymes 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase and 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase. In vitro tests ruled out the possibility that the tolerance was due to altered forms of these enzymes. Families showed significant variability with regard to EPSP and DAHP synthase levels, measured at different stages during seedling growth; however, not even these traits were correlated with in vivo response to glyphosate. The possible role of other physiological processes in determining the increased tolerance to the herbicide is discussed.

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.

5-enol-Pyruvyl-Shikimate-3-Phosphate Synthase from Zea mays Cultured Cells (Purification and Properties).

The shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase (3-phosphoshikimate-1-carboxyvinyl transferase, EC 2.5.1.19) was purified from cultured maize (Zea mays L. var Black Mexican Sweet) cells. Homogeneous enzyme preparations were obtained by a four-step procedure using ammonium sulfate fractionation, anion- and cation-exchange chromatography, and substrate elution from a cellulose phosphate column. The last step resulted in two well-separated activities of about the same molecular weight. A 2000- to 3000-fold purification, with an overall recovery of one-fourth of the initial activity, was achieved. Both EPSP synthase isoforms were characterized with respect to structural, kinetic, and biochemical properties. Only slight differences are seen in molecular mass, activation energy, and apparent affinities for the two substrates. A more pronounced difference was found between their thermal inactivation rates. Two EPSP synthase isoforms were also elucidated in crude homogenates by anion-exchange fast protein liquid chromatography. This allowed us to follow their expression during a culture growth cycle. One form was found at substantial levels throughout, whereas the other increased in exponentially growing cells and declined in late-logarithmic phase. The analysis of highly purified plastid preparations demonstrated a plastidial localization of both proteins. Possible functional roles for maize EPSP synthase isozymes, with regard to the dual-pathway hypothesis and to the recent findings on defense-related aromatic biosynthesis in higher plants, are discussed.

Hybrid formation in the shikimate pathway enzyme dehydroquinase

The guanidine hydrochloride-induced denaturation of unmodified and product-linked forms of the dimeric shikimate pathway enzyme 3-dehydroquinase are shown, by fluorescence spectroscopy, to be reversible. Mixing the unfolded polypeptides prior to renaturation yields a hybrid dimer, which has been identified by isoelectric focussing and its relative yield quantitated by laser densitometry.

Identification of the essential histidine residue at the active site of Escherichia coli dehydroquinase.

The shikimate pathway enzyme 3-dehydroquinase is very susceptible to inactivation by the group-specific reagent diethyl pyrocarbonate (DEP). Inactivation follows pseudo first-order kinetics and exhibits a second-order rate constant of 148.5 M-1 min-1. An equilibrium mixture of substrate and product substantially protects against inactivation by DEP, suggesting that residues within the active site are being modified. Complete inactivation of the enzyme correlates with the modification of 6 histidine residues/subunit as determined by difference spectroscopy at 240 nm. Enzymic activity can be restored by hydroxylamine treatment, which is also consistent with the modification occurring at histidine residues. Using the kinetic method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1558), it was shown that modification of a single histidine residue leads to inactivation. Ligand protection experiments also indicated that 1 histidine residue was protected from DEP modification. pH studies show that the pKa for this inactivation is 6.18, which is identical to the single pKa determined from the pH/log Vmax profile for the enzyme. A single active site peptide was identified by differential peptide mapping in the presence and absence of ligand. This peptide was found to comprise residues 141-158; of the 2 histidines in this peptide (His-143 and His-146), only one, His-143, is conserved among all type I dehydroquinases. We propose that His-143 is the active site histidine responsible for DEP-mediated inactivation of dehydroquinase and is a good candidate for the general base that has been postulated to participate in the mechanism of this enzyme.

The Mycobacterium tuberculosis shikimate pathway genes: Evolutionary relationship between biosynthetic and catabolic 3-dehydroquinases

The Mycobacterium tuberculosis shikimate pathway genes designated aroB and aroQ encoding 3-dehydroquinate synthase and 3-dehydroquinase, respectively were isolated by molecular cloning and their nucleotide sequences determined. The deduced dehydroquinate synthase amino acid sequence from M. tuberculosis showed high similarity to those of equivalent enzymes from prokaryotes and filamentous fungi. Surprisingly, the deduced M. tuberculosis 3-dehydroquinase amino acid sequence showed no similarity to other characterised prokaryotic biosynthetic 3-dehydroquinases (bDHQases). A high degree of similarity was observed, however, to the fungal catabolic 3-dehydroquinases (cDHQases) which are active in the quinic acid utilisation pathway and are isozymes of the fungal bDHQases. This finding indicates a common ancestral origin for genes encoding the catabolic dehydroquinases of fungi and the biosynthetic dehydroquinases present in some prokaryotes. Deletion of genes encoding shikimate pathway enzymes represents a possible approach to generation of rationally attenuated strains of M. tuberculosis for use as live vaccines.

Stabilization of the shikimate pathway enzyme dehydroquinase by covalently bound ligand

Reversible binding of a ligand to an enzyme active site can elicit a variety of changes in the protein, such as conformational changes (close to the site of binding or communicated over long distances), changes in the ionization state of surrounding amino acid side chains, changes in the interaction of the target protein with other subunits (or other proteins), or even changes in the thermodynamic stability of the protein. Relatively little attention has been given to studying these effects in proteins to which the ligand has been irreversibly bound, yet this can be a convenient way of studying the effects of ligand binding in the absence of association/dissociation equilibria. We report the dramatic changes which occur to the shikimate pathway enzyme dehydroquinase when ligand is attached to its active site after borohydride reduction of the mechanistically important Schiff's base intermediates. The effects of this modification have been characterized by limited proteolysis, circular dichroism, guanidine hydrochloride denaturation, and differential scanning calorimetry. The conclusions from these studies are that although anchoring the ligand at the active site does not cause a gross change in conformation, it does increase markedly the conformational stability of the protein. This is conclusively established by three separate experiments: 1) the modified protein is completely resistant to proteases, whereas the unmodified protein is very susceptible to proteolysis; 2) the concentration of guanidine hydrochloride required to unfold the ligand-linked dehydroquinase is 3-4-fold greater than that of the unmodified protein; 3) the melting temperature (Tm) of the modified protein is 40 degrees C higher than that of the unmodified protein. These results are a very clear example of the thermodynamic link between ligand binding, conformational stability, and proteolytic susceptibility in vitro and will be a useful system for dissecting the contributions of individual protein-ligand interactions to these parameters.