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At least 523 records · Page 29Linked to original sources
PubMed2006

Structure of Arabidopsis dehydroquinate dehydratase-shikimate dehydrogenase and implications for metabolic channeling in the shikimate pathway.

The bifunctional enzyme dehydroquinate dehydratase-shikimate dehydrogenase (DHQ-SDH) catalyzes the dehydration of dehydroquinate to dehydroshikimate and the reduction of dehydroshikimate to shikimate in the shikimate pathway. We report the first crystal structure of Arabidopsis DHQ-SDH with shikimate bound at the SDH site and tartrate at the DHQ site. The interactions observed in the DHQ-tartrate complex reveal a conserved mode for substrate binding between the plant and microbial DHQ dehydratase family of enzymes. The SDH-shikimate complex provides the first direct evidence of the role of active site residues in the catalytic mechanism. Site-directed mutagenesis and mechanistic analysis revealed that Asp 423 and Lys 385 are key catalytic groups and Ser 336 is a key binding group. The arrangement of the two functional domains reveals that the control of metabolic flux through the shikimate pathway is achieved by increasing the effective concentration of dehydroshikimate through the proximity of the two sites.

Alcohol Oxidoreductases↗
PubMed1996

Binding of the oxidized, reduced, and radical flavin species to chorismate synthase. An investigation by spectrophotometry, fluorimetry, and electron paramagnetic resonance and electron nuclear double resonance spectroscopy.

Chorismate synthase (EC 4.6.1.4) binds oxidized riboflavin-5'-phosphate mononucleotide (FMN) with a KD of 30 microM at 25 degrees C, but in the presence of 5-enolpyruvylshikimate-3-phosphate (EPSP), the KD decreases to ca. 20 nM. Similar effects occur with the substrate analogue (6R)-6-fluoro-EPSP (KD = 36 nM) and chorismate (KD = 540 nM). Fluorescence of oxidized FMN is slightly quenched in the presence of chorismate synthase. Addition of EPSP or the (6R)-6-fluoro analogue causes a shift of the fluorescence from 520 to 495 nm. Chorismate causes no shift in, but a quenching of, the fluorescence emission maximum. In the presence of EPSP, (6R)-6-fluoro-EPSP, or chorismate, the neutral flavinsemiquinone is generated. The electron paramagnetic resonance (EPR) line width of the flavin radical is indicative of a neutral flavinsemiquinone. Frozen solution electron nuclear double resonance (ENDOR) of the radical with (6R)-6-fluoro-EPSP shows a number of proton ENDOR line pairs. The largest splitting is assigned to a hyperfine coupling to the methyl group beta-protons at position 8 of the isoalloxazine ring. The hyperfine-coupling (hfc) components have values of A perpendicular = 8.07 MHz and A parallel = 9.60 MHz, giving Aiso of 8.58 MHz, consistent with a neutral semiquinone form. The isotropic hfc coupling of the 8-methyl protons with (6R)-6-fluoro-EPSP decreases by about 0.5 MHz when chorismate is bound, indicating that the spin density distribution within the isoalloxazine ring system depends critically on the nature of the ligand. The redox potential of FMN in the presence of chorismate synthase was 95 mV more positive than that of free FMN (at pH 7.0), equivalent to a 1660-fold tighter binding of reduced FMN. The pH dependence of the redox potential of chorismate synthase-bound FMN exhibits a slope of -30 mV per pH unit between pH 6 and 9, indicating that the two-electron reduction of the flavin is associated with the uptake of one proton; this, and the UV-visible spectrum, is consistent with the reduced flavin being bound to chorismate synthase in its monoanionic form.

Electron Spin Resonance Spectroscopy↗
PubMed1996

An EPSP synthase inhibitor joining shikimate 3-phosphate with glyphosate: synthesis and ligand binding studies.

A novel EPSP synthase inhibitor 4 has been designed and synthesized to probe the configurational details of glyphosate recognition in its herbicidal ternary complex with enzyme and shikimate 3-phosphate (S3P). A kinetic evaluation of the new 3-dephospho analog 12, as well as calorimetric and (31)P NMR spectroscopic studies of enzyme-bound 4, now provides a more precise quantitative definition for the molecular interactions of 4 with this enzyme. The very poor binding, relative to 4, displayed by the 3-dephospho analog 12 is indicative that 4 has a specific interaction with the S3P site. A comparison of Ki(calc) for 12 versus the Ki(app) for 4 indicates that the 3-phosphate group in 4 contributes about 4.8 kcal/mol to binding. This compares well with the 5.2 kcal/mol which the 3-phosphate group in S3P contributes to binding. Isothermal titration calorimetry demonstrates that 4 binds to free enzyme with an observed Kd of 0.53 +/- 0.04 microM. As such, 4 binds only 3-fold weaker than glyphosate and about 150-fold better than N-methylglyphosate. Consequently, 4 represents the most potent N-alkylglyphosate derivative identified to date. However, the resulting thermodynamic binding parameters clearly demonstrate that the formation of EPSPS x 4 is entropy driven like S3P. The binding characteristics of 4 are fully consistent with a primary interaction localized at the S3P subsite. Furthermore, (31)P NMR studies of enzyme-bound 4 confirm the expected interaction at the shikimate 3-phosphate site. However, the chemical shift observed for the phosphonate signal of EPSPS x 4 is in the opposite direction than that observed previously when glyphosate binds with enzyme and S3P. Therefore, when 4 occupies the S3P binding site, there is incomplete overlap at the glyphosate phosphonate subsite. As a glyphosate analog inhibitor, the potency of 4 most likely arises from predominant interactions which occur outside the normal glyphosate binding site. Consequently, 4 is best described as an S3P-based substrate-analog inhibitor. These combined results corroborate the previous kinetic model [Gruys, K. J., Marzabadi, M. R., Pansegrau, P. D., & Sikorski, J. A. (1993) Arch. Biochem. Biophys. 304, 345-351], which suggested that 4 interacts well with the S3P subsite but has little, if any, interaction at the expected glyphosate phosphonate or phosphoenolpyruvate-Pi subsites.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1996

Ligand geometry of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase from rotational-echo double-resonance NMR.

The 46-kDa enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP. The reaction is inhibited by N-(phosphonomethyl)glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. As part of a solid-state NMR characterization of this structure, we have used dipolar recovery at the magic angle (DRAMA) and rotational-echo double resonance (REDOR) to determine intra- and interligand internuclear distances. DRAMA was used to determine the single 31P-31P distance, while REDOR was used to determine one 31P-15N distance and five 31P-13C distances. These experimental distances were used as restraints in molecular dynamics simulations of an S3P-Glp complex to examine the geometry of the two ligands relative to one another in the ternary complex. The simulations were compared to unrestrained simulations of the EPSP synthase tetrahedral intermediate and its phosphonate analog. The results suggest that Glp is unlikely to bind in the same fashion as PEP, a conclusion that is consistent with recent studies that have questioned the role of Glp as a transition-state or intermediate analog.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1996

Analysis of fluoromethyl group chirality establishes a common stereochemical course for the enolpyruvyl transfers catalyzed by EPSP synthase and UDP-GlcNAc enolpyruvyl transferase.

The stereochemistry of transient methyl group formation at C-3 of phosphoenolpyruvate (PEP) in the reaction catalyzed by 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase has been examined using the pseudosubstrates, (E)- and (Z)-3-fluorophosphoenolpyruvate (FPEP). Kinetically stable, chiral [1H, 2H]fluoromethyl analogs of the reaction tetrahedral intermediate were isolated and subjected to decomposition and stereochemical analysis. EPSP synthase was found to catalyze the 2-re face addition of solvent-derived hydrogen to C-3 of FPEP (corresponding to the 2-si face of PEP). Comparison of these data with prior analogous work on the MurA reaction [Kim, D.H., Lees, W.J., & Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380-6381] suggests that the two enolpyruvyl transferases share a common stereochemical course, further strengthening the mechanistic, structural, and evolutionary relationship between the two enzymes.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1999

Substrate and inhibitor-induced conformational changes in the structurally related enzymes UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS).

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) have both a unique three-dimensional topology and overall reaction mechanism in common. In the case of MurA, the substrate-free, unliganded protein exhibits an "open" conformation. Upon binding of substrates, the protein forms a much more tightly packed so-called "closed" form following an induced fit mechanism. In this closed form, the substrates are properly positioned for catalysis. On the basis of the structural and mechanistic similarities of MurA and EPSPS, a similar conformational change is likely to occur in EPSPS to generate a catalytically competent active site. However, there is currently little experimental evidence available to support the occurrence of such a conformational change in EPSPS. Using limited tryptic digestion of MurA,(1) it could be shown that formation of the "closed" conformation of MurA is accompanied by a marked increase of stability toward proteolytic degradation. Formation of the closed conformation was achieved by addition of either an excess of both substrates or the sugar nucleotide substrate in conjunction with the antibiotic fosfomycin. Analysis of the MurA tryptic fragments by MALDI-TOF mass spectrometry demonstrates that the protection of the protein in either case is caused by (1) a specific shielding of regions thereby becoming less accessible as a result of the conformational change, and (2) an unspecific overall protection of the whole protein due to an apparently reduced flexibility of the peptide backbone in the binary and ternary complexes. The establishment of methods to describe the effects of tryptic digestion on MurA under various conditions was then extended to EPSPS. Although EPSPS was found to be much more stable toward proteolysis than MurA, the presence of shikimate 3-phosphate (S3P) and the inhibitor glyphosate led to a pronounced suppression of proteolytic degradation. When unliganded EPSPS was treated with trypsin, three of the peptide fragments obtained could be identified by mass spectrometry. Two of these are located in a region corresponding to the "catalytic" loop in MurA which participates in the conformational change. This indicates a conformational change in EPSPS, similar to the one observed in MurA, leading to the protection mentioned above. Corroborating evidence was obtained using a conformational sensitive monoclonal antibody against EPSPS which showed a 20-fold reduced affinity toward the protein complexed with S3P and glyphosate as compared to the unliganded enzyme.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMedDate not supplied

MFA for overdetermined systems reviewed and compared with rna expression data to elucidate the difference in shikimate yield between carbon- and phosphate-limited continuous cultures of E. coli W3110.shik1.

The present contribution focuses on the mathematical techniques used to solve steady state metabolic models for the case of an overdetermined system. Even when parts of the system are underdetermined it is possible to solve the model partially and obtain statistically meaningful results. This is illustrated with data gathered from a set of E. coli W3110.shik1 phosphate- or carbon-limited continuous cultures. It is shown that the low yield in shikimate for C-limited cultures is not due to a lower flux going to the shikimate pathway but is caused by a high secretion of byproducts. Carbon-limited cultures could be better for shikimate production than carbon-abundant cultures provided the byproduct secretion is reduced. Finally, flux calculations are compared with RNA expression data.

Carbon↗
PubMed2004

Creation of a shikimate pathway variant.

The competition between the Escherichia coli carbohydrate phosphotransferase system and 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase for phosphoenolpyruvate limits the concentration and yield of natural products microbially synthesized via the shikimate pathway. To circumvent this competition for phosphoenolpyruvate, a shikimate pathway variant has been created. 2-Keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases encoded by Escherichia coli dgoA and Klebsiella pneumoniae dgoA are subjected to directed evolution. The evolved KDPGal aldolase isozymes exhibit 4-8-fold higher specific activities relative to that for native KDPGal aldolase with respect to catalyzing the condensation of pyruvate and d-erythrose 4-phosphate to produce DAHP. To probe the ability of the created shikimate pathway variant to support microbial growth and metabolism, growth rates and synthesis of 3-dehydroshikimate are examined for E. coli constructs that lack phosphoenolpruvate-based DAHP synthase activity and rely on evolved KDPGal aldolase for biosynthesis of shikimate pathway intermediates and products.

Aldehyde-Lyases↗
PubMed2003

Shikimate accumulates in both glyphosate-sensitive and glyphosate-resistant horseweed (Conyza canadensis L. Cronq.).

Horseweed (Conyza canadensis) is a cosmopolitan weed that commonly grows throughout North America. Horseweed that is not completely controlled by normal applications of glyphosate has been reported in western Tennessee. This research had three objectives: (1) to develop and validate an analytical procedure for the quantitative determination of shikimate, an important indicator of glyphosate activity in plants; (2) to confirm resistance to glyphosate in a horseweed population; and (3) to examine the accumulation of shikimate in both glyphosate-resistant and glyphosate-susceptible horseweed plants. The analytical procedure to determine shikimate used extraction with 1 M HCl for 24 h, followed by liquid chromatography using photodiode array detection, and shikimate recoveries were >or=82%. Glyphosate applications of both 0.84 kg ae/ha (the standard application rate) and 3.8 kg ae/ha to susceptible plants caused complete plant death. The same glyphosate applications to putative resistant populations caused less than 15% growth reduction as determined by visual evaluations, and fresh weights of these resistant plants 17 days after glyphosate treatment (DAT) were reduced an average of 45% in one population and were not affected in a different population. This direct comparison conclusively confirms that horseweed plants collected in western Tennessee in 2002 are resistant to 4 times the normal application dosage of glyphosate. The glyphosate-resistant horseweed biotypes still exhibited some herbicidal effects from the glyphosate, such as yellowing in the most actively growing, apical shoot meristems. The yellowing in the shoot apexes was transitory, and the plants recovered from this damage. Shikimate concentrations in all untreated horseweed plants were less than 100 microg/g, which was significantly less than that in all plants which had been treated with 0.84 kg ae/ha of glyphosate. Unexpectedly, shikimate accumulated (>1000 microg/g) in both resistant populations and the susceptible population. However, there were differences in shikimate accumulation patterns between resistant and susceptible horseweed biotypes. Shikimate concentrations in resistant populations declined about 40% from 2 to 4 DAT, while shikimate concentrations in the susceptible horseweed plants increased about 35% from 2 to 4 DAT. The confirmed resistance of a widespread weed implies that alternative control strategies for glyphosate-resistant horseweed will be needed in those no-tillage production systems where it commonly occurs.

Conyza↗
PubMed2003

Aroma composition of Vitis vinifera Cv. tannat: the typical red wine from Uruguay.

The free volatiles, as well as those released from the glycosidically bound forms by enzyme hydrolysis, have been analyzed to chracterize young Tannat wines from two successive vintages. The Tannat wine showed some aroma profile peculiarities detected in the free forms but, above all, in the bound fraction for the level and profile of the norisoprenoidic fraction. Among the free volatile compunds, a rather low content of C(6) alcohols with a prevalence of cis-3-hexen-1-ol on the trans form and sometimes a remarkable level of trans-2-hexen-1-ol seem to be typical for the variety. C(13)-norisoprenoidic and monoterpenic volatiles made up approximately 42% of the total level of the volatiles observed in the glycosidase enzyme-released fraction. The other volatiles were C(6) alcohols (6%) and benzenoid compounds (51%). The dominating monoterpene alcohols were the cis and trans isomers of 3,7-dimethyl-1,6-octadiene-3,8-diol (8-hydroxylinalool). The C(13)-norisoprenoid pattern was composed by 3-hydroxy-beta-damascone, 3-oxo-alpha-ionol, vomifoliol, 4-oxo-beta-ionol, 3-oxo-7,8-dihydro-alpha-ionol, 4-oxo-7,8-dihydro-beta-ionol, grasshopper ketone, and 7,8-dihydrovomifoliol.

Chromatography, Gas↗
PubMed2002

Regioselective enzymatic acylation of methyl shikimate. Influence of acyl chain length and solvent polarity on enzyme specificity.

Candida antarctica lipase A (CAL-A) selectively catalyzes the acylation at the secondary C-4 hydroxyl group of methyl shikimate (2), which possesses three secondary hydroxyl groups, the C-3 allylic one being chemically more reactive. The effect both of the acyl group of the acylating agents and of the solvent polarity has been studied. The selectivity of CAL-A is almost complete with acyl donors that possess short chains. However, when acyl donors have longer chains, better results are obtained by C. antarctica lipase B (CAL-B).

Acylation↗
PubMed2004

Enzymatic synthesis of a ring-contracted analogue of 5-enolpyruvylshikimate-3-phosphate.

[reaction: see text] Three ring-contracted mimics of shikimate-3-phosphate, formed from the triols by shikimate kinase, were evaluated as substrates of the next enzyme in the pathway, EPSP synthase. The cyclopentylidene analogue (+)-2P was converted enzymatically to the enolpyruvyl derivative, thus demonstrating the second step of an artificial biosynthetic sequence.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1998

Effects of sample preparation conditions on biomolecular solid-state NMR lineshapes.

Sample preparation conditions with the 46 kDa enzyme complex of 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, shikimate-3-phosphate (S3P) and glyphosate (GLP) have been examined in an attempt to reduce linewidths in solid-state NMR spectra. The linewidths of 31P resonances associated with enzyme bound S3P and GLP in the lyophilized ternary complex have been reduced to 150 +/- 12 Hz and 125 +/- 7 Hz respectively, by a variety of methods involving additives and freezing techniques.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗