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SCHEME 1. Proposed reaction mechanisms for DAHP synthase. Path A, stepwise mechanism for the formation of a linear hemiketal bisphosphate intermediate. Path B, the hemiketal intermediate formation takes place through an oxocarbonium intermediate (concerted mechanism).
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Escherichia coli phenylalanine-sensitive 3-deoxy-arabino-heptulosonate 7-phosphate synthase (DAHP synthase) catalyzes the net aldol condensation of phosphoenolpyruvate and erythrose 4-phosphate to form 3-deoxy-D-arabino-heptulosonate 7-phosphate and inorganic phosphate. For the first time, the presteady-state kinetic analysis of the Phe-sensitive D...
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Context 1
... Dependence of k cat on MnCl 2 Concentration-Manganese has been reported as the metal ion that gives the highest steady-state rate (19). In the present study, the K d (1 M) and k cat (23 s 1 ) values were determined by following the consump- tion of PEP in a continuous spectrophotometric assay (Fig. 1). The k cat value was calculated based on protein concentration by assuming a concentration of the active sites equal to protein concentration. However, the results of burst experiments with the Mn-DAHP synthase (see below and Fig. 2) show that only 30% of the enzyme is active. Therefore, the corrected k cat value based upon the ...Context 2
... based on protein concentration by assuming a concentration of the active sites equal to protein concentration. However, the results of burst experiments with the Mn-DAHP synthase (see below and Fig. 2) show that only 30% of the enzyme is active. Therefore, the corrected k cat value based upon the concentration of the active sites is 75 s 1 (Fig. 1). This value is in accordance with the previously reported rate determined for the enzyme ...Context 3
... the other hand, Zn(II) is, in general, tetrahedral or distorted tetrahedral, although other geometries have been noted and have been reviewed recently (21). The crystal structures of Mn-DAHP synthase (10, 22) show a distorted octahedral coor- dination geometry for Mn(II), with one of the ligands being the thiol group of Cys-61. Cysteine is a very unlikely ligand for Mn(II) because none of the manganese-containing enzymes currently present in the Protein Data Bank contain a cysteine coordinated to Mn(II). ...Citations
... The first stage is the accumulation of erythrose-4-phosphate and phosphoenolpyruvate from the pentose phosphate and glycolysis pathways, resulting in the production of 3-Deoxy-D-arabinose-heptulosonate-7-phosphate and inorganic phosphate, respectively. DAHP Synthase catalyzes this reaction, which is a crucial stage in the process (Furdui et al. 2004). In plants, lignin production is done by two enzymes that methylate cinnamic acid to produce hydroxylate and monolignol derivatives. ...
Plantain (Plantago major L.) is a valuable medicinal plant that contains a high level of secondary metabolites. The objective of this research is to examine the oxidative stress, such as drought and heavy metals, on the production of these metabolites in liquid medium. Drought stress was induced by using polyethylene glycol 6000 at three levels of 0%, 6%, and 12%. Heavy metal stress was induced by using HgCl2 at three levels of 0, 4 and 8 µM on 21-day -old plants immersed in liquid MS medium. Sampling was performed at 0, 2, 4, and 6 days after stress from treated and control plants. The Total content of secondary metabolites, including phenol, flavonoid, terpene, alkaloid, and anthocyanin, was evaluated using spectrophotometry methods. Additionally, the correlation between the levels of these metabolites and the expression of crucial genes involved in their biosynthesis, such as phenylalanine ammonia-lyase synthesize (PAL), 3-deoxy-D-arabino-heptulosonate-7-phosphate-synthase (DAHPS), 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoAR), caffeic acid o-methyl transferase (COMT), and squalene epoxidase (SQE), was examined using Real-Time PCR. The results of the variance analysis for phenol, flavonoid, terpene, alkaloid, and anthocyanin, which were affected by PEG and HgCl2, showed significant changes in secondary metabolites in treated plants compared to control plants. Although the patterns of gene expression and secondary metabolite production did not always follow the same pattern, the effect of stresses on increasing the expression of some genes related to phenol and terpenoid production pathway was significant.
... 22 The metal ion plays a structural role by coordinating key residues to help form an active site catalytic motif, which seems to favor binding of a water molecule for activation. 23 In the native state, Mn(II) is hexacoordinated to the side chains of an aspartyl (Asp 441 ), glutamyl (Glu 411 ), histidyl (His 369 ), and cysteinyl (Cys 87 ), with a water molecule filling the sixth position. Although DAHPS is a transferase, its active site arrangement resembles the motif of many nonheme transitionmetal-dependent oxygenases and sulfoxidases that aid oxyfunctionalization of carbon and C−S activation of organic substrates. ...
The shikimate pathway, which produces aromatic amino acids and key intermediates, is critical to the viability of the tuberculosis-causing pathogen Mycobacterium tuberculosis. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) catalyzes the first committed step of this pathway and possesses regulatory functions. Its active site contains two cysteinyls: one (Cys87) bound to a metal ion, while the other (Cys440) is in proximity to the first but is located on a connecting loop. This arrangement seemingly appeared as a disulfide linkage. However, Cys440 is not metal binding, and its positioning indicates that it could collapse the disulfide linkage. Hence, its potential role may be more than simply structural support of the active site fold. Using a multiscale computational approach, molecular dynamics (MD) simulations, and DFT-based calculations, the influence of Cys440 on the active site properties has been investigated. MD simulations reveal an unusually long disulfide bond, more than 3.0 Å, whereas DFT calculations identified two stable active site conformers in the triplet and quintet spin states. Analysis of group spin density distribution identified antiferromagnetic coupling in each conformer, which suggests their relatively low potential energy and stable conformations. The conformer in the triplet spin state could favor enzyme reactivity due to its low HOMO-LUMO energy gap. In addition, reduction of the Cys440 thiolate group results in collapse of the active site metal-ligand configuration with large exothermicity. Hence, Cys440 could activate and inactivate the enzyme. For the first time, the study revealed the role of Cys440 as being vital for the catalytic activity of the enzyme rather than solely for the structural stabilization of its active site. Thus, the findings may lead to a novel basis for antituberculosis drug design and development that would disrupt the contributions of the Cys440.
... A variety of metals can activate DAHP synthases, including Fe 2+ and Mn 2+ . Note that the metal-coordinating glutamate can sometimes bind the metal in bidentate mode, changing the overall geometry to an octahedral shape (Shumilin et al., 1999;Furdui et al., 2004;König et al., 2004;Shumilin et al., 2004). ...
Apart from oxygenic photosynthesis, the extent of manganese utilization in bacteria varies from species to species and also appears to depend on external conditions. This observation is in striking contrast to iron, which is similar to manganese but essential for the vast majority of bacteria. To adequately explain the role of manganese in pathogens, we first present in this review that the accumulation of molecular oxygen in the Earth’s atmosphere was a key event that linked manganese utilization to iron utilization and put pressure on the use of manganese in general. We devote a large part of our contribution to explanation of how molecular oxygen interferes with iron so that it enhances oxidative stress in cells, and how bacteria have learned to control the concentration of free iron in the cytosol. The functioning of iron in the presence of molecular oxygen serves as a springboard for a fundamental understanding of why manganese is so valued by bacterial pathogens. The bulk of this review addresses how manganese can replace iron in enzymes. Redox-active enzymes must cope with the higher redox potential of manganese compared to iron. Therefore, specific manganese-dependent isoenzymes have evolved that either lower the redox potential of the bound metal or use a stronger oxidant. In contrast, redox-inactive enzymes can exchange the metal directly within the individual active site, so no isoenzymes are required. It appears that in the physiological context, only redox-inactive mononuclear or dinuclear enzymes are capable of replacing iron with manganese within the same active site. In both cases, cytosolic conditions play an important role in the selection of the metal used. In conclusion, we summarize both well-characterized and less-studied mechanisms of the tug-of-war for manganese between host and pathogen.
... DAH7PS initiates the shikimate pathway for the biosynthesis of aromatic amino acids (Phe, Tyr, and Trp), by catalyzing the reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) to produce DAH7P. This reaction is dependent on the presence of a divalent metal ion, such as Mn 2+ , Cd 2+ and Zn 2+ (14,15). Allosteric regulation of this gateway enzyme tunes the output of the shikimate pathway to cellular requirements (6)(7)(8). ...
Modular protein assembly has been widely reported as a mechanism for constructing allosteric machinery. Recently, a distinctive allosteric system has been identified in a bi-enzyme assembly comprising a 3-deoxy-D-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM). These enzymes catalyze the first and branch point reactions of aromatic amino acid biosynthesis in the bacterium Prevotella nigrescens (PniDAH7PS), respectively. The interactions between these two distinct catalytic domains support functional inter-reliance within this bifunctional enzyme. The binding of prephenate, the product of CM-catalyzed reaction, to the CM domain is associated with a striking rearrangement of overall protein conformation that alters the interdomain interactions and allosterically inhibits the DAH7PS activity. Here, we have further investigated the complex allosteric communication demonstrated by this bifunctional enzyme. We observed allosteric activation of CM activity in the presence of all DAH7PS substrates. Using small angle X-ray scattering (SAXS) experiments we show that changes in overall protein conformations and dynamics are associated with the presence of different DAH7PS substrates and the allosteric inhibitor prephenate. Furthermore, we have identified an extended interhelix loop located in CM domain, loopC320-F333, as a crucial segment for the interdomain structural and catalytic communications. Our results suggest that the dual-function enzyme PniDAH7PS contains a reciprocal allosteric system between the two enzymatic moieties as a result of this bidirectional interdomain communication. This arrangement allows for a complex feedback and feedforward system for control of pathway flux by connecting the initiation and branch point of aromatic amino acid biosynthesis.
... The activity of EDTA-treated apo MtDAHPS could be restored in assays containing Co 2+ , Mn 2+ , Cd 2+ , Cu 2+ , Zn 2+ and Ca 2+ , with Co 2+ and Mn 2+ yielding the best results for restoring activity [28]. Furdui and coworkers [32] conducted experiments demonstrating that, in the case of E. coli DAHPS, the metal ion in the active site of the enzyme seems to play a structural role, orchestrating the arrangement of the active site residues in a position favorable for water activation. In this view, the geometry coordination of different metal ions may be a valuable factor for rational inhibitor design. ...
Roughly a third of the world’s population is estimated to have latent Mycobacterium tuberculosis infection, being at risk of developing active tuberculosis (TB) during their lifetime. Given the inefficacy of prophylactic measures and the increase of drug-resistant M. tuberculosis strains, there is a clear and urgent need for the development of new and more efficient chemotherapeutic agents, with selective toxicity, to be implemented on patient treatment. The component enzymes of the shikimate pathway, which is essential in mycobacteria and absent in humans, stand as attractive and potential targets for the development of new drugs to treat TB. This review gives an update on published work on the enzymes of the shikimate pathway and some insight on what can be potentially explored towards selective drug development.
... This was perhaps not surprising considering the conformational flexibility of the unliganded protein observed by HDX (see below) and the known connection between highly flexible peptides and susceptibility to aggregation, 40−42 as well as the fact that even freshly purified DAHPS(Phe) was previously found to be only 30% active. 43 Protein inactivation during the titration might be expected to alter the apparent K d values; however, the fitted K d values were close to those determined by other methods, with K d,Mn = 5.3 ± 1.7 μM and K d,DAHP oxime = 3.1 ± 1.2 μM. ...
... are significantly higher than most values reported previously for E. coli DAHPS(Phe),33,43,47 though the K M (1s) values derived from the single substrate Michaelis−Menten equation matched well. Using the correct K M values is important insofar as the apparent K i value depends on the K M values used in eq 3. Using the K M (1s) values in eq 3 gave an apparent K i value of 7 nM for DAHP oxime, 200-fold lower than the correct value of 1.5 ± 0.4 μM. ...
... Activities of G6PD (ZWF1), 6PGD (Gnd1), ATP-PRTase (His1), and DAHP synthase (Aro3,4) were assessed using previously published kinetic spectrophotometric assays (83)(84)(85). Assays used to identify or confirm previously unrecognized regulation are detailed below. Putative regulators of each enzyme were adjusted to the pH of the reaction buffer. ...
Quantitation of metabolic pathway regulation
Although metabolic biochemical pathways are well understood, less is known about precisely how reaction rates or fluxes through the various enzymes are controlled. Hackett et al. developed a method to quantitate such regulatory influence in yeast. They monitored concentrations of metabolites, enzymes, and potential regulators by LC-MS/MS (liquid chromatography–tandem mass spectrometry) and isotope ratio measurements for 56 reactions, over 100 metabolites, and 370 metabolic enzymes in yeast in 25 different steady-state conditions. Bayesian analysis was used to examine the probability of regulatory interactions. Regulation of flux through the pathways was predominantly controlled by changes in the concentration of small-molecule metabolites rather than changes in enzyme abundance. The analysis also revealed previously unrecognized regulation between pathways.
Science , this issue p. 432
... Continuous assay of purified DAHP synthase. Purified DAHP synthase was assayed as described previously (27). The purified enzyme was diluted 4-fold, thus reducing EDTA levels to 0.25 mM, and treated with 50 M tris(2-carboxyethyl)phosphine (TCEP) and 0.75 mM each metal in 0.1 M KP i (pH 6.4) under anoxic conditions for 10 min at ambient temperature. ...
In Escherichia coli aromatic biosynthesis is the process that has shown the greatest sensitivity to hydrogen peroxide stress. This pathway has long been recognized to be sensitive to superoxide as well, but the molecular target was unknown. Feeding experiments indicated that the bottleneck lies early in the pathway, and the suppressive effects of fur mutations and manganese supplementation suggested the involvement of a metalloprotein. The 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHP synthase) activity catalyzes the first step in the pathway, and it is provided by three isozymes known to rely upon a divalent metal. This activity progressively declined when cells were stressed with either oxidant. The purified enzyme was activated more strongly by ferrous iron than by other metals, and only this metalloform could be inactivated by hydrogen peroxide or superoxide. We infer that iron is the prosthetic metal in vivo. Both oxidants displace the iron atom from the enzyme. In peroxide-stressed cells the enzyme accumulated as an apoprotein, potentially with an oxidized cysteine residue. In superoxide-stressed cells the enzyme acquired a non-activating zinc ion in its active site, an apparent consequence of the repeated ejection of iron. Manganese supplementation protected the activity in both cases, which matches the ability of manganese to metallate the enzyme and to provide substantial oxidant-resistant activity. DAHP synthase thus belongs to a family of mononuclear iron enzymes that are disabled by oxidative stress. To date all the intracellular injuries caused by physiological doses of these reactive oxygen species have arisen from the oxidation of reduced iron centers.
... AroF and AroH are as homodimers (McCandliss et al., 1978; Shumilin et al., 1999). Different studies have demonstrated that the N-terminal of DAHP synthase involves in the formation of a putative inhibitor-binding site (Wagner et al., 2000; Furdui et al., 2004). Jossek et al. (2001) reported that substitution of Asn8 to Lys8 resulted AroF to be L-Tyr insensitive. ...
3-Deoxy-D-arabino-heptulosonate 7-phosphate synthases (DAHP synthases) encoded by aroF is the first enzyme of the shikimate pathway. In the present work, an AroF variant with deficiency of the residue Ile11 (named AroF*) was characterized to be L-Tyr insensitive. According to three-dimensional structure analysis, nine AroF variants were constructed with truncation of different N-terminal fragments, and overexpression of the variants AroF(Δ(1-9)) , AroF(Δ(1-10)) , AroF(Δ(1-12)) especially AroF(Δ(1-11)) significantly increased the accumulation of L-Phe. However, the AroG and AroH variants with similar truncations of the N-terminal fragments decreased the production of L-Phe. By co-overexpressing AroF(Δ(1-11)) and PheA(fbr) , the production of L-Phe was increased from 2.36±0.07 g L(-1) (co-overexpression of the wild-type AroF and PheA(fbr) ) to 4.29±0.06 g L(-1) . The novel variant AroF(Δ(1-11)) showed great potential for the production of aromatic amino acids and their derivatives. This article is protected by copyright. All rights reserved.
... [73][74] An alternative mechanism has been proposed in the literature where attack of water or hydroxyl at C2 PEP precedes carbon-carbon bond formation between PEP and E4P. 85 This proposal is problematic due to the following reasons: (1) it requires electrophilic reactivity at the C2 PEP which is unprecedented. (2) The product of such a step would be a carbanion at C3 PEP or in case of a concerted reaction, carbanionic character of C3 PEP in the transition state. ...
... Type I DAH7PS enzymes are generally smaller, with molecular weights below 40 kDa, as opposed to the Type II enzymes which usually have a monomeric molecular weight of around 54 kDa. The type Iα DAH7PS enzymes include the well-characterised enzymes from E. coli 75,77,85,[87][88] and Saccharomyces cerevisiae. 78,89 The type Iα enzymes share little sequence homology with the DAH7PS enzymes from the type Iβ subfamily, which include the enzymes from Pyroccocus furiosus, 80,82,90 Thermotoga maritima, 76,[91][92] Bacillus subtilis [93][94] and Listeria monocytogenes. ...
The shikimate pathway is responsible for the biosynthesis of the aromatic amino acids and other
aromatic metabolites in plants, micro-organisms and apicomplexan parasites. The shikimate
pathway is essential in bacteria and plants, but absent from mammals, which has led to interest
in the enzymes of the pathway as targets for the design of antimicrobial and herbicidal agents.
3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed
step of the shikimate pathway, the condensation of phosphoenolpyruvate and erythrose 4-
phosphate to yield 3-deoxy-D-arabino-heptulosonate 7-phosphate. The subject of this thesis is
the investigation of inhibition and allosteric regulation of the DAH7PS enzyme from Mycobacterium
tuberculosis (MtuDAH7PS), the pathogen that causes tuberculosis. Tuberculosis
remains a major health threat to the global community, and the emergence of multi-drug
resistant strains highlights the need for new tuberculosis treatments. Inhibitors of MtuDAH7PS
have the potential to be developed into new anti-tuberculosis drugs.
Chapter 2 describes the design, synthesis and evaluation of active site inhibitors based on
intermediate mimic scaffolds. The intermediate mimics synthesised represent the first reported
example of inhibitors targeting the active site of MtuDAH7PS. The most active compounds
tested displayed inhibition constants in the sub-micromolar range, making them the most potent
inhibitors of any DAH7PS enzyme reported to date.
MtuDAH7PS displays a complex and subtle mechanism of synergistic regulation: The enzyme
is inhibited by binary combinations of the aromatic amino acids tryptophan (Trp), phenylalanine
(Phe) and tyrosine (Tyr). Three allosteric binding sites were identified using X-ray crystallographic
analysis of MtuDAH7PS in complex with Trp and Phe. While these crystal structures
led to the identification of an allosteric binding site which preferentially binds Trp, the role and
selectivity of the other two sites with respect to Phe and Tyr remained unclear. The results
described in Chapter 3 provide structural and biochemical evidence for the hypothesis that each
of the three allosteric binding sites has a preference for binding one of the aromatic amino acids
Trp, Phe and Tyr, respectively. The results furthermore show that the ternary combination of
Trp, Phe and Tyr synergistically regulates MtuDAH7PS, leading to almost complete loss of
enzymatic activity in the presence of all three allosteric ligands.
In Chapter 4, the interaction of MtuDAH7PS with the naturally less common D-enantiomers of
the aromatic amino acids is described. It was found that the D-enantiomers of the aromatic
amino acids have no effect on enzymatic activity of MtuDAH7PS, suggesting an efficient
iv
mechanism by which the enzyme can discriminate between allosteric ligands of opposite
configuration. Studies of the binding affinity of the D-amino acids to MtuDAH7PS as well as
structural characterisation of MtuDAH7PS-D-amino acid complexes by X-ray crystallographic
analysis suggest that D-Trp and D-Phe can still bind to their respective sites. The lack of
inhibition is attributed to subtle differences in the binding mode of the D-enantiomers of the
ligands compared to the L-enantiomers.
Chapter 5 details the discovery of alternative ligands and inhibitors targeting the allosteric sites
of MtuDAH7PS using virtual screening. Libraries of potential alternative ligands were docked
into the allosteric sites of MtuDAH7PS and the predicted docking poses were used to guide the
selection of compounds for physical screening. Using this approach, a number of ligands and
inhibitors of MtuDAH7PS were discovered and their interaction with the enzyme structurally
characterised. Comparison of the crystallographically observed binding modes of new ligands
with the docking poses predicted by computational docking highlighted potential improvements
to the virtual screening method. The analysis of the correlation between ligand binding modes
and inhibition of enzymatic activity provided further insight into which interactions between the
allosteric ligand and the binding site are crucial in order to achieve inhibition. The crystal
structures of MtuDAH7PS in complex with the new alternative ligands can serve as a starting
point for the design of ligands with increased affinity and potency.
