Targeting the role of a key conserved motif for substrate selection and catalysis by 3-deoxy-D-manno-octulosonate 8-phosphate synthase

3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between three-carbon phosphoenolpyruvate (PEP) and five-carbon d-arabinose 5-phosphate (A5P), generating KDO8P, a key intermediate in the biosynthetic pathway to 3-deoxy-D-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Both metal-dependent and metal-independent forms of KDO8PS have been characterized. KDO8PS is evolutionarily and mechanistically related to the first enzyme of the shikimate pathway, the obligately divalent metal ion-dependent 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) that couples PEP and four-carbon D-erythrose 4-phosphate (E4P) to give DAH7P. In KDO8PS, an absolutely conserved KANRS motif forms part of the A5P binding site, whereas in DAH7PS, an absolutely conserved KPR(S/T) motif accommodates E4P. Here, we have characterized four mutants of this motif (AANRS, KAARS, KARS, and KPRS) in metal-dependent KDO8PS from Acidithiobacillus ferrooxidans and metal-independent KDO8PS from Neisseria meningitidis to test the roles of the universal Lys and the Ala-Asn portion of the KANRS motif. The X-ray structures, determined for the N. meningitidis KDO8PS mutants, indicated no gross structural penalty resulting from mutation, but the subtle changes observed in the active sites of these mutant proteins correlated with their altered catalytic function. (1) The AANRS mutations destroyed catalytic activity. (2) The KAARS mutations lowered substrate selectivity, as well as activity. (3) Replacing KANRS with KARS or KPRS destroyed KDO8PS activity but did not produce a functional DAH7PS. Thus, Lys is critical to catalysis, and other changes are necessary to switch substrate specificity for both the metal-independent and metal-dependent forms of these enzymes.     

Expression, purification, and characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Pyrococcus furiosus

The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P). DAH7PS from the hyperthermophile Pyrococcus furiosus has been expressed in Escherichia coli. The expressed protein was insoluble but was partially solubilized as a dimer by the inclusion of 200 mM KCl in the cell lysis buffer. An effective two step purification procedure has been developed. The first step resulted in a high degree of purification and involved lysis by sonication at approximately 40 degrees C followed by a heat treatment at 70 degrees C. A continuous assay measuring the loss of PEP at 232 nm at elevated temperatures was also developed. Temperature, pH, and divalent metal ions all had an effect on the extinction coefficient of PEP. Purified recombinant P. furiosus DAH7PS is a dimer with a subunit Mr of 29,226 (determined by ESMS), shows resistance to denaturation by SDS, has activity over a broad pH range, and has an activation energy of 88 kJmol-1. The kinetic parameters are Km (PEP) 120 microM, Km (E4P) 28 microM, and kcat 1.5s-1, at 60 degrees C and pH 6.8. DAH7PS is not inhibited by phenylalanine, tyrosine, or tryptophan. EDTA inactivates the enzyme and enzyme activity is restored by a wide range of divalent metal ions including (in order of decreasing effectiveness): Zn2+, Cd2+, Mn2+, Co2+, Ni2+, Ca2+, Hg2+, and Cu2+. This detailed characterization of the DAH7PS from P. furiosus raises the possibility that the subfamily Ibeta DAH7PS enzymes are metal ion dependent, contrary to previous predictions.     

Substrate ambiguity and crystal structure of Pyrococcus furiosus 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase: an ancestral 3-deoxyald-2-ulosonate-phosphate synthase?

3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAH7PS) catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and the four-carbon monosaccharide D-erythrose 4-phosphate (E4P). DAH7PS from the hyperthermophile Pyrococcus furiosus is a member of the DAH7PS Ibeta subfamily, which also includes the KDO8PS enzymes. KDO8PS (3-deoxy-D-manno-octulosonate-8-phosphate synthase) catalyzes a closely related reaction of PEP with the five-carbon monosaccharide D-arabinose 5-phosphate (A5P). DAH7PS from P. furiosus requires a metal ion for activity and, unlike other characterized DAH7PS enzymes, is not inhibited by aromatic amino acids. Purified P. furiosus DAH7PS is able to utilize not only the four-carbon phosphorylated monosaccharides E4P and 2-deoxy-D-erythrose 4-phosphate but also the five-carbon phosphorylated monosaccharides A5P, D-ribose 5-phosphate, and 2-deoxy-D-ribose 5-phosphate with similar kcat but much increased KM values. DL-glyceraldehyde 3-phosphate and D-glucose 6-phosphate are not substrates. The structure of recombinant P. furiosus DAH7PS in complex with PEP was determined to 2.25 A resolution. The asymmetric unit consists of a dimer of (beta/alpha)8-barrel subunits. Analysis of the buried surfaces formed by dimerization and tetramerization, as observed in the crystal structure, provides insight into both the oligomeric status in solution and the substrate ambiguity of P. furiosus DAH7PS. P. furiosus DAH7PS is both the first archaeal and the first "naked" DAH7PS (without N-terminal extensions) to be fully characterized functionally and structurally. The broad substrate specificity of this DAH7PS, the lack of allosteric inhibition, and various structural features indicate that, of the enzymes characterized to date, P. furiosus DAH7PS may be the contemporary protein closest to the ancestral type I enzyme.     

Synthesis and antibacterial activity of mechanism-based inhibitors of KDO8P synthase and DAH7P synthase

KDO8PS (3-deoxy-D-manno-2-octulosonate-8-phosphate synthase) and DAH7PS (3-deoxy-D-arabino-2-heptulosonate-7-phosphate synthase) are attractive targets for the development of new anti-infectious agents. Both enzymes appear to proceed via a common mechanism involving the reaction of phosphoenolpyruvate (PEP) with arabinose 5-phosphate or erythrose-4-phosphate, to produce the corresponding ulosonic acids, KDO8P and DAH7P, respectively. The synthesis of new inhibitors closely related to the supposed tetrahedral intermediate substrates for the enzymes is described. The examination of the antibacterial activity of these derivatives is reported.     

The structure of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase from Mycobacterium tuberculosis reveals a common catalytic scaffold and ancestry for type I and type II enzymes

The shikimate pathway, responsible for the biosynthesis of aromatic compounds, is essential for the growth of Mycobacterium tuberculosis and is a potential target for the design of new anti-tuberculosis drugs. The first step of this pathway is catalyzed by 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS). The DAH7PSs have been classified into two apparently unrelated types and, whereas structural data have been obtained for the type I DAH7PSs, no structural information is available for their type II counterparts. The type II DAH7PS from M.tuberculosis has been expressed in Escherichia coli, purified, functionally characterized and crystallized. It is found to be metal ion-dependent and subject to feedback inhibition by phenylalanine, tryptophan, tyrosine and chorismate, with a significant synergistic effect when tryptophan is used in combination with phenylalanine. The crystal structure of M.tuberculosis DAH7PS has been determined by single-wavelength anomalous diffraction and refined at 2.3A in complex with substrate phosphoenolpyruvate and Mn(2+). The structure reveals a tightly associated dimer of (beta/alpha)(8) TIM barrels. The monomer fold, the arrangement of key residues in the active site, and the binding modes of PEP and Mn(2+), all match those of the type I enzymes, and indicate a common ancestry for the type I and type II DAH7PSs, despite their minimal sequence identity. In contrast, the structural elements that decorate the core (beta/alpha)(8) fold differ from those in the type I enzymes, consistent with their different regulatory and oligomeric properties.     

Reciprocal allostery arising from a bienzyme assembly controls aromatic amino acid biosynthesis in Prevotella nigrescens

Modular protein assembly has been widely reported as a mechanism for constructing allosteric machinery. Recently, a distinctive allosteric system has been identified in a bienzyme 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 interreliance 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, loop_C320-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.     

Investigating the role of the hydroxyl groups of substrate erythrose 4-phosphate in the reaction catalysed by the first enzyme of the shikimate pathway

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthase catalyses the first step of the shikimate pathway, which is responsible for the biosynthesis of aromatic amino acids in microorganisms and plants. This enzyme catalyses an aldol reaction between phosphoenolpyruvate and D-erythrose 4-phosphate to generate DAH7P. Both 2-deoxyerythrose 4-phosphate and 3-deoxyerythrose 4-phosphate were synthesised and tested as alternative substrates for the enzyme. Both compounds were found to be substrates for the DAH7P synthases from Escherichia coli, Pyrococcus furiosus and Mycobacterium tuberculosis, consistent with an acyclic mechanism for the enzyme for which neither C2 nor C3 hydroxyl groups are required for catalysis. The enzymes all showed greater tolerance for the loss of the C2 hydroxyl group than the C3 hydroxyl group.     

Substrate deactivation of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase by erythrose 4-phosphate.

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS, EC 4.1.2.15) catalyzes the condensation of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to give DAH7P via an ordered sequential mechanism. In the absence of PEP (the first substrate to bind), E4P binds covalently to the phenylalanine-sensitive DAH7PS of Escherichia coli, DAH7PS(Phe), deactivating the enzyme. Activity is restored on addition of excess PEP but not if deactivation was carried out in the presence of sodium cyanoborohydride. Electrospray mass spectrometry indicates that a single E4P is bound to the protein. These data are consistent with a slow, reversible Schiff base reaction of the aldehydic functionality of E4P with a buried lysine. Molecular modeling indicates that Lys186, a residue at the base of the substrate-binding cavity involved in hydrogen bonding with PEP, is well placed to react with E4P forming an imine linkage that is substantially protected from solvent water.     

Complex Formation between Two Biosynthetic Enzymes Modifies the Allosteric Regulatory Properties of Both: AN EXAMPLE OF MOLECULAR SYMBIOSIS*

Allostery, where remote ligand binding alters protein function, is essential for the control of metabolism. Here, we have identified a highly sophisticated allosteric response that allows complex control of the pathway for aromatic amino acid biosynthesis in the pathogen Mycobacterium tuberculosis. This response is mediated by an enzyme complex formed by two pathway enzymes: chorismate mutase (CM) and 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS). Whereas both enzymes are active in isolation, the catalytic activity of both enzymes is enhanced, and in particular that of the much smaller CM is greatly enhanced (by 120-fold), by formation of a hetero-octameric complex between CM and DAH7PS. Moreover, on complex formation M. tuberculosis CM, which has no allosteric response on its own, acquires allosteric behavior to facilitate its own regulatory needs by directly appropriating and partly reconfiguring the allosteric machinery that provides a synergistic allosteric response in DAH7PS. Kinetic and analytical ultracentrifugation experiments demonstrate that allosteric binding of phenylalanine specifically promotes hetero-octameric complex dissociation, with concomitant reduction of CM activity. Together, DAH7PS and CM from M. tuberculosis provide exquisite control of aromatic amino acid biosynthesis, not only controlling flux into the start of the pathway, but also directing the pathway intermediate chorismate into either Phe/Tyr or Trp biosynthesis.     

Synergistic Allostery,a Sophisticated Regulatory Network for the Control of Aromatic Amino Acid Biosynthesis in Mycobacterium tuberculosis

The shikimate pathway, responsible for aromatic amino acid biosynthesis, is required for the growth of Mycobacterium tuberculosis and is a potential drug target. The first reaction is catalyzed by 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS). Feedback regulation of DAH7PS activity by aromatic amino acids controls shikimate pathway flux. Whereas Mycobacterium tuberculosis DAH7PS (MtuDAH7PS) is not inhibited by the addition of Phe, Tyr, or Trp alone, combinations cause significant loss of enzyme activity. In the presence of 200 μm Phe, only 2.4 μm Trp is required to reduce enzymic activity to 50%. Reaction kinetics were analyzed in the presence of inhibitory concentrations of Trp/Phe or Trp/Tyr. In the absence of inhibitors, the enzyme follows Michaelis-Menten kinetics with respect to substrate erythrose 4-phosphate (E4P), whereas the addition of inhibitor combinations caused significant homotropic cooperativity with respect to E4P, with Hill coefficients of 3.3 (Trp/Phe) and 2.8 (Trp/Tyr). Structures of MtuDAH7PS/Trp/Phe, MtuDAH7PS/Trp, and MtuDAH7PS/Phe complexes were determined. The MtuDAH7PS/Trp/Phe homotetramer binds four Trp and six Phe molecules. Binding sites for both aromatic amino acids are formed by accessory elements to the core DAH7PS (β/α)₈ barrel that are unique to the type II DAH7PS family and contribute to the tight dimer and tetramer interfaces. A comparison of the liganded and unliganded MtuDAH7PS structures reveals changes in the interface areas associated with inhibitor binding and a small displacement of the E4P binding loop. These studies uncover a previously unrecognized mode of control for the branched pathways of aromatic amino acid biosynthesis involving synergistic inhibition by specific pairs of pathway end products.     

Synthesis and evaluation of dual site inhibitors of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAH7P) synthase catalyses the first step of the shikimate pathway for the biosynthesis of aromatic compounds. Enzymes of this pathway have been identified as potential targets for drug design. The reaction catalysed by DAH7P synthase is an aldol condensation between phosphoenolpyruvate (PEP) and d-erythrose 4-phosphate (E4P). In this study inhibitors of DAH7P synthase were prepared which were designed to fit into the binding sites of both PEP and E4P substrates simultaneously. Inhibitors, known to target the PEP binding site, were extended using a C4 linker to include an appropriately placed phosphate group in order to access the phosphate-binding site of E4P. A small increase in inhibition was observed with this modification, and the inhibition results have been rationalised by induced-fit docking.     

The Functional Unit of Neisseria meningitidis 3-Deoxy-?-Arabino-Heptulosonate 7-Phosphate Synthase Is Dimeric

Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (NmeDAH7PS) adopts a homotetrameric structure consisting of an extensive and a less extensive interface. Perturbation of the less extensive interface through a single mutation of a salt bridge (Arg126-Glu27) formed at the tetramer interface of all chains resulted in a dimeric DAH7PS in solution, as determined by small angle X-ray scattering, analytical ultracentrifugation and analytical size-exclusion chromatography. The dimeric NmeDAH7PS^R126S variant was shown to be catalytically active in the aldol-like condensation reaction between d-erythrose 4-phosphate and phosphoenolpyruvate, and allosterically inhibited by l-phenylalanine to the same extent as the wild-type enzyme. The dimeric NmeDAH7PS^R126S variant exhibited a slight reduction in thermal stability by differential scanning calorimetry experiments and a slow loss of activity over time compared to the wild-type enzyme. Although NmeDAH7PS^R126S crystallised as a tetramer, like the wild-type enzyme, structural asymmetry at the less extensive interface was observed consistent with its destabilisation. The tetrameric association enabled by this Arg126-Glu27 salt-bridge appears to contribute solely to the stability of the protein, ultimately revealing that the functional unit of NmeDAH7PS is dimeric.     

Dynamic cross-talk among remote binding sites: the molecular basis for unusual synergistic allostery

Allosteric regulation of protein function is critical for metabolic control. Binding of allosteric effectors elicits a functional change in a remote ligand binding site on a protein by altering the equilibrium between different forms in the protein ensemble. 3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step in the shikimate pathway, which is responsible for the biosynthesis of aromatic amino acids Trp, Phe, and Tyr. Feedback regulation by the aromatic amino acids is important for controlling the cellular levels of the aromatic amino acids, and many organisms have two or more DAH7PS isozymes that show differing sensitivities to aromatic compounds. Mycobacterium tuberculosis expresses a single DAH7PS that is insensitive to the presence of a single amino acid yet shows extraordinary synergistic inhibition by combinations of the pathway end products Trp and Phe. The Trp+Phe-bound structure for M. tuberculosis DAH7PS, showing two separate binding sites occupied by Trp and Phe for each monomer of the tetrameric protein, was obtained by cocrystallization. Comparison of this structure with the ligand-free M. tuberculosis DAH7PS demonstrates that there is no significant change in conformation upon ligand binding, suggesting that contributions from altered dynamic properties of the enzyme may account for the allosteric inhibition. Isothermal titration calorimetry experiments demonstrate that the inhibitor binding sites are in direct communication. Molecular dynamics simulations reveal different changes in dynamic fluctuations upon single ligand binding compared to dual ligand binding. These changes account for the cross-talk between inhibitor binding sites and the active site, simultaneously potentiating both dual ligand binding and diminution of catalytic function.     

Potent inhibitors of a shikimate pathway enzyme from Mycobacterium tuberculosis: combining mechanism- and modeling-based design

Tuberculosis remains a serious global health threat, with the emergence of multidrug-resistant strains highlighting the urgent need for novel antituberculosis drugs. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the shikimate pathway for the biosynthesis of aromatic compounds. This pathway has been shown to be essential in Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. DAH7PS catalyzes a condensation reaction between P-enolpyruvate and erythrose 4-phosphate to give 3-deoxy-d-arabino-heptulosonate 7-phosphate. The enzyme reaction mechanism is proposed to include a tetrahedral intermediate, which is formed by attack of an active site water on the central carbon of P-enolpyruvate during the course of the reaction. Molecular modeling of this intermediate into the active site reported in this study shows a configurational preference consistent with water attack from the re face of P-enolpyruvate. Based on this model, we designed and synthesized an inhibitor of DAH7PS that mimics this reaction intermediate. Both enantiomers of this intermediate mimic were potent inhibitors of M. tuberculosis DAH7PS, with inhibitory constants in the nanomolar range. The crystal structure of the DAH7PS-inhibitor complex was solved to 2.35 Å. Both the position of the inhibitor and the conformational changes of active site residues observed in this structure correspond closely to the predictions from the intermediate modeling. This structure also identifies a water molecule that is located in the appropriate position to attack the re face of P-enolpyruvate during the course of the reaction, allowing the catalytic mechanism for this enzyme to be clearly defined.     

A radiometric assay for 5-enolpyruvylshikimate-3-phosphate synthase

A new assay for 5-enolpyruvylshikimate-3-phosphate synthase is described. This enzyme of the shikimate pathway of aromatic amino acid biosynthesis generates 5-enolpyruvylshikimate 3-phosphate and orthophosphate from phosphoenolpyruvate and shikimate 3-phosphate. The shikimate pathway is present in bacteria and plants but not in mammals. The assay employs a paper-chromatographic separation of radiolabeled substrate from product. The method is specific, is sensitive to 50 pmol of product, and is suitable for use in crude extracts of bacteria. This enzyme appears to be the primary target site of the commercial herbicide glyphosate (N-phosphonomethyl glycine). A procedure for the enzymatic synthesis of [¹⁴C]shikimate 3-phosphate from the commercially available precursor [¹⁴C]shikimic acid is also described.     

An extended β7α7 substrate-binding loop is essential for efficient catalysis by 3-deoxy-D-manno-octulosonate 8-phosphate synthase

The enzyme 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the reaction between phosphoenolpyruvate and arabinose 5-phosphate (A5P) in the first committed step in the biosynthetic pathway for the formation of 3-deoxy-D-manno-octulosonate, an important component in the cell wall of Gram-negative bacteria. KDO8P synthase is evolutionarily related to the first enzyme of the shikimate pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthase, which uses erythrose 4-phosphate in place of A5P. The A5P binding site in KDO8P synthase is formed by three long loops that extend from the core catalytic (β/α)(8) barrel, β2α2, β7α7, and β8α8. The extended β7α7 loop is always present in KDO8P synthase yet is not observed for DAH7P synthase. Modeling of this loop indicated interactions between this loop and the extended β2α2 loop; both loops provide key hydrogen-bonding contacts with A5P. The two absolutely conserved residues on the β7α7 loop (Gln and Ser) were mutated to Ala in both the metal-dependent KDO8P synthase from Acidithiobacillus ferrooxidans and the metal-independent KDO8P synthase from Neisseria meningitidis. In addition, mutants were constructed for both enzymes with the extended β7α7 loop excised to match the DAH7P synthase architecture. Removal of the loop extension severely hindered efficient catalysis, dramatically increasing the K(m)(A5P) and reducing the k(cat) for both enzymes. Excision of the complete loop was far more detrimental to catalysis than the double mutations of the two conserved Gln and Ser residues. Therefore, the presence of the entire extended β7α7 loop is important for efficient catalysis by KDO8P synthase, with the loop acting to promote efficient and productive binding of A5P.     

Allosteric inhibitor specificity of Thermotoga maritima 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase

3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first step of the shikimate pathway for the biosynthesis of aromatic amino acids. Allosteric regulation of Thermotoga maritima DAH7PS is mediated by l-Tyr binding to a discrete ACT regulatory domain appended to a core catalytic (β/α)₈ barrel. Variants of T. maritima DAH7PS (TmaDAH7PS) were created to probe the role of key residues in inhibitor selection. Substitution Ser31Gly severely reduced inhibition by l-Tyr. In contrast both l-Tyr and l-Phe inhibited the TmaHis29Ala variant, while the variant where Ser31 and His29 were interchanged (His29Ser/Ser31His), was inhibited to a greater extent by l-Phe than l-Tyr. These studies highlight the role and importance of His29 and Ser31 for determining both inhibitory ligand selectivity and the potency of allosteric response by TmaDAH7PS.     

Mode of action of glyphosate in Candida maltosa

The broad-spectrum herbicide glyphosate inhibits the growth of Candida maltosa and causes the accumulation of shikimic acid and shikimate-3-phosphate. Glyphosate is a potent inhibitor of three enzymes of aromatic amino acid biosynthesis in this yeast. In relation to tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and dehydroquinate synthase, the inhibitory effect appears at concentrations in the mM range, but 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase is inhibited by micromolar concentrations of glyphosate. Inhibition of partially purified EPSP synthase reaction by glyphosate is competitive with respect to phosphoenolpyruvate (PEP) with a K _i -value of 12 M. The app. K _m for PEP is about 5-fold higher and was 62 M. Furthermore, the presence of glyphosate leads to derepression of many amino acid biosynthetic enzymes.Abbreviations DAHP 3-deoxy-D-arabino-heptulosonate 7-phosphate - EPSP synthase 5-enolpyruvylshikimate 3-phosphate synthase - PEP phosphoenolpyruvate - S-3-P shikimate-3-phosphate     

Destabilization of the Homotetrameric Assembly of 3-Deoxy-d-Arabino-Heptulosonate-7-Phosphate Synthase from the Hyperthermophile Pyrococcus furiosus Enhances Enzymatic Activity

Many proteins adopt homomeric quaternary structures to support their biological function, including the first enzyme of the shikimate pathway that is ultimately responsible for the biosynthesis of the aromatic amino acids in plants and microorganisms. This enzyme, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAH7PS), adopts a variety of different quaternary structures depending on the organism in which it is found. The DAH7PS from the hyperthermophilic archaebacterium Pyrococcus furiosus was previously shown to be tetrameric in its crystalline form, and this quaternary association is confirmed in an improved structure in a different crystal system. This tetramer is also present in solution as revealed by small-angle X-ray scattering and analytical ultracentrifugation. This homotetrameric form has two distinct interfaces, both of which bury over 10% each of the surface area of a single monomer. Substitution of Ile for Asp in the hydrophobic region of one interface gives a protein with a remarkable 4-fold higher maximum catalytic rate than the wild-type enzyme. Analytical ultracentrifugation at pH 7.5 reveals that the tetrameric form is destabilized; although the protein crystallizes as a tetramer, equilibrium exists between tetrameric and dimeric forms with a dissociation constant of 22 μM. Thus, under the conditions of kinetic assay, the enzyme is primarily dimeric, revealing that the dimeric form is a fully functional catalyst. However, in comparison to the wild-type protein, the thermal stability of the dimeric protein is significantly compromised. Thus, an unusual compromise of enzymatic activity versus stability is observed for this DAH7PS from an organism that favors a hyperthermophilic environment.     

Structure and mechanism of 3-deoxy-D-manno-octulosonate 8-phosphate synthase

3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate. KDO8P is the phosphorylated precursor of 3-deoxy-D-manno-octulosonate, an essential sugar of the lipopolysaccharide of Gram-negative bacteria. The crystal structure of the Escherichia coli KDO8P synthase has been determined by multiple wavelength anomalous diffraction and the model has been refined to 2.4 A (R-factor, 19.9%; R-free, 23.9%). KDO8P synthase is a homotetramer in which each monomer has the fold of a (beta/alpha)(8) barrel. On the basis of the features of the active site, PEP and A5P are predicted to bind with their phosphate moieties 13 A apart such that KDO8P synthesis would proceed via a linear intermediate. A reaction similar to KDO8P synthesis, the condensation of phosphoenolpyruvate, and erythrose 4-phosphate to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P), is catalyzed by DAH7P synthase. In the active site of DAH7P synthase the two substrates PEP and erythrose 4-phosphate appear to bind in a configuration similar to that proposed for PEP and A5P in the active site of KDO8P synthase. This observation suggests that KDO8P synthase and DAH7P synthase evolved from a common ancestor and that they adopt the same catalytic strategy.