Characterization of a new feedback-resistant 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase AroF of Escherichia coli

Tyrosine feedback-inhibits the 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase isoenzyme AroF of Escherichia coli. Here we show that an Asn-8 to Lys-8 substitution in AroF leads to a tyrosine-insensitive DAHP synthase. This mutant enzyme exhibited similar activities (v=30-40 U mg(-1)) and substrate affinities (K(m)(erythrose-4-phosphate)=0.5 mM, positive cooperativity with respect to phospho(enol)pyruvate) as the wild-type AroF, but showed decreased thermostability. An engineered AroF enzyme lacking the seven N-terminal residues also was tyrosine-resistant. These results strongly suggest that the N-terminus of AroF is involved in the molecular interactions occurring in the feedback-inhibition mechanism.     

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase. Purification, properties, and kinetics of the tyrosine-sensitive isoenzyme from Escherichia coli.

The tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate lyase (pyruvate-phosphorylating), EC 4.2.1.15) was purified to homogeneity from extracts of Escherichia coli K12. A spectrophotometric assay of the enzyme activity, based on the absorption difference of substrates and products at 232 nm, was developed. The enzyme has a molecular weight of 66,000 as judged by gel filtration on Sephadex G-200, and a subunit molecular weight of 39,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This suggests either a rapid monomer-dimer equilibrium, or a very asymmetric shape for the native enzyme. The enzyme shows a narrow pH optimum around pH 7.0. The enzyme is stable for several months when stored at -20 degrees in phosphate buffer containing phosphoenol-pyruvate. Intersecting lines in double reciprocal plots of initial velocity data at substrate concentrations in the micromolar range suggest a sequential mechanism with-catalyzed reaction. Product inhibition studies specify an ordered sequential BiBi mechanism with a dead-end E-P complex. The feedback inhibitor tyrosine at concentrations above 10 muM exhibits noncompetitive inhibition with respect to erythrose-4-P, and competitive inhibition with respect to the other substrate, P-enolpyruvate. In addition, tyrosine at concentrations of at least 10 muM causes an alteration of one or more than one kinetic parameter of the enzyme.     

The nucleotide sequence of the aroF gene of Escherichia coli and the amino acid sequence of the encoded protein, the tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

The translated sequence of aroF, the first structural gene of the tyrosine operon of Escherichia coli, has been determined. The 1068 nucleotides encode the 356 amino acids that form the subunit of the dimeric tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase. The primary structure of this enzyme has been confirmed by automated Edman degradation of peptide fragments produced by cleavage with cyanogen bromide, limited trypsin digestion, Staphylococcus aureus strain V8 protease, or mild acid hydrolysis. The amino acid sequence of this enzyme is compared with the sequence of the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, deduced from the aroG DNA sequence (Davies, W. D., and Davidson, B. E. (1982) Nucleic Acids Res. 10, 4045-4058).     

Evolution of the regulatory isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase present in the Escherichia coli genealogy.

The evolutionary history of isozymes for 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase has been constructed in a phylogenetic cluster of procaryotes (superfamily B) that includes Escherichia coli. Members of superfamily B that have been positioned on a phylogenetic tree by oligonucleotide cataloging possess one or more of four distinct isozymes of DAHP synthase. DAHP synthase-0 is insensitive to feedback inhibition, while DAHP synthase-Tyr, DAHP synthase-Trp, and DAHP synthase-Phe are sensitive to feedback inhibition by L-tyrosine, L-tryptophan, and L-phenylalanine, respectively. The evolutionary history of this isozyme family can be deduced within superfamily B by using a cladistic methodology of maximum parsimony (R. A. Jensen, Mol. Biol. Evol. 2:92-108, 1985). DAHP synthase-0 was found in Acinetobacter species and in Oceanospirillum minutulum, organisms that also possess DAHP synthase-Tyr. These two isozymes were apparently present in a common ancestor that predated the evolutionary divergence of contemporary superfamily B sublineages. DAHP synthase-0 is postulated to have been the evolutionary forerunner of DAHP synthase-Trp. The newly evolved DAHP synthase-Trp is postulated to have possessed sensitivity to feedback inhibition by chorismate as well as by L-tryptophan, chorismate sensitivity having been retained in rRNA group I pseudomonads (minor sensitivity), group V pseudomonads (very sensitive), and Lysobacter enzymogenes (ultrasensitive). Organisms constituting the enteric lineage of the phylogenetic tree (including a cluster of four Oceanospirillum species) have all lost the chorismate sensitivity of DAHP synthase-Trp. The absence of DAHP synthase-Phe in the Oceanospirillum cluster of organisms supports the previous conclusion that DAHP synthase-Phe evolved recently within superfamily B, being present only Escherichia coli and its close relatives.     

Redox regulation of Arabidopsis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

The cDNA for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Arabidopsis encodes a polypeptide with an amino-terminal signal sequence for plastid import. A cDNA fragment encoding the processed form of the enzyme was expressed in Escherichia coli. The resulting protein was purified to electrophoretic homogeneity. The enzyme requires Mn(2+) and reduced thioredoxin (TRX) for activity. Spinach (Spinacia oleracea) TRX f has an apparent dissociation constant for the enzyme of about 0.2 microM. The corresponding constant for TRX m is orders of magnitude higher. In the absence of TRX, dithiothreitol partially activates the enzyme. Upon alkylation of the enzyme with iodoacetamide, the dependence on a reducing agent is lost. These results indicate that the first enzyme in the shikimate pathway of Arabidopsis appears to be regulated by the ferredoxin/TRX redox control of the chloroplast.     

Insights into the mechanism of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (Phe) from Escherichia coli using a transient kinetic analysis

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 DAHP synthase from E. coli is reported. The steady-state and presteady-state kinetic parameters of the DAHP synthase reconstituted with Mn(II), Cu(II), and Zn(II) were compared. These studies showed the following: 1) product release is rate-limiting for all of the three metal ions studied under physiologically relevant conditions; 2) concentration of the active sites of the metal-containing DAHP synthase is increasing from Mn- (30%) to Zn- (52%) and to Cu-DAHP synthase (88%); 3) rate constant for product formation is higher in Mn- (130-200 s(-1)) than Cu- (55 s(-1)) and Zn-DAHP synthase (6.8 s(-1)); and 4) steady-state rate (rate constant for product release) is higher for the Mn- (70 s(-1)) than for Cu- (5.6 s(-1)) and Zn-DAHP synthase (1.8 s(-1)). In addition, an examination of the reaction kinetics at lower pH reveals that for Cu-DAHP synthase, product release is no longer rate-limiting, whereas the Mn- and Zn-DAHP synthase show a slower rate of product formation, suggesting that the intermediate formation becomes rate-limiting in product formation. Also, a deuterium-isotope effect on the burst rate constant of product formation for Mn-DAHP synthase was observed at pH 6.0. This supports the hypothesis that the role of metal ion in E. coli DAHP synthase is to position the amino acids with the appropriate geometry required to coordinate and activate the water molecule.     

Structure and characterization of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Aeropyrum pernix

The first enzyme in the shikimic acid biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), varies significantly in size and complexity in the bacteria and plants that express it. The DAH7PS from the archaebacterium Aeropyrum pernix (DAH7PS(Ap)) is among the smallest and least complex of the DAH7PS enzymes, leading to the hypothesis that DAH7PS(Ap) would not be subject to feedback regulation by shikimic acid pathway products. We overexpressed DAH7PS(Ap) in Escherichia coli, purified it, and characterized its enzymatic activity. We then solved its X-ray crystal structure with a divalent manganese ion and phosphoenolpyruvate bound (PDB ID: 1VS1). DAH7PS(Ap) is a homodimeric metalloenzyme in solution. Its enzymatic activity increases dramatically above 60 °C, with optimum activity at 95 °C. Its pH optimum at 60 °C is 5.7. DAH7PS(Ap) follows Michaelis-Menten kinetics at 60 °C, with a K(M) for erythrose 4-phosphate of 280 μM, a K(M) for phosphoenolpyruvate of 891 μM, and a k(cat) of 1.0 s(-1). None of the downstream products of the shikimate biosynthetic pathway we tested inhibited the activity of DAH7PS(Ap). The structure of DAH7PS(Ap) is similar to the structures of DAH7PS from Thermatoga maritima (PDB ID: 3PG8) and Pyrococcus furiosus (PDB ID: 1ZCO), and is consistent with its designation as an unregulated DAH7PS.     

A cDNA encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Solanum tuberosum L

A cDNA encoding potato (Solanum tuberosum L.) 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway, was cloned into phage lambda gt11. The clone represents the first cDNA for this enzyme from any eukaryotic source. The nucleotide sequence of the cDNA was determined, and its identity was confirmed through partial amino acid sequence analysis of the encoded enzyme. The cDNA contains a 1527-base pair open reading frame that encodes a polypeptide with a calculated molecular weight of 56,153. The amino terminus of the deduced polypeptide resembles a chloroplast transit sequence. Amino acid sequence identities between the mature potato enzyme and the homologous isoenzymes from Escherichia coli are only about 22%. The potato cDNA hybridized to various plant mRNAs that are all about 2 kilobases in size.     

Stereospecific deuteration of 2-deoxyerythrose 4-phosphate using 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

Racemic 2-deoxyerythrose 4-phosphate was synthesized and one enantiomer of this compound was found to be a substrate for Escherichia coli 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway. When the reaction was carried out in deuterium oxide, an enzyme-catalyzed regio- and stereoselective incorporation of deuterium into the product was observed.     

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.     

Crystal structure of phenylalanine-regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

BACKGROUND: In microorganisms and plants the first step in the common pathway leading to the biosynthesis of aromatic compounds is the stereospecific condensation of phosphoenolpyruvate (PEP) and D-erythrose-4-phosphate (E4P) giving rise to 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP). This reaction is catalyzed by DAHP synthase (DAHPS), a metal-activated enzyme, which in microorganisms is the target for negative-feedback regulation by pathway intermediates or by end products. In Escherichia coli there are three DAHPS isoforms, each specifically inhibited by one of the three aromatic amino acids. RESULTS: The crystal structure of the phenylalanine-regulated form of DAHPS complexed with PEP and Pb2+ (DAHPS(Phe)-PEP-Pb) was determined by multiple wavelength anomalous dispersion phasing utilizing the anomalous scattering of Pb2+. The tetramer consists of two tight dimers. The monomers of the tight dimer are coupled by extensive interactions including a pair of three-stranded, intersubunit beta sheets. The monomer (350 residues) is a (beta/alpha)8 barrel with several additional beta strands and alpha helices. The PEP and Pb2+ are at the C-ends of the beta strands of the barrel, as is SO4(2-), inferred to occupy the position of the phosphate of E4P. Mutations that reduce feedback inhibition cluster about a cavity near the twofold axis of the tight dimer and are centered approximately 15 A from the active site, indicating the location of a separate regulatory site. CONCLUSIONS: The crystal structure of DAHPS(Phe)-PEP-Pb reveals the active site of this key enzyme of aromatic biosynthesis and indicates the probable site of inhibitor binding. This is the first reported structure of a DAHPS; the structure of its two paralogs and of a variety of orthologs should now be readily determined by molecular replacement.     

Some kinetic properties of the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Neurospora crassa.

The steady-state kinetic properties of purified tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Neurospora crassa were examined. The results suggest that the enzyme obeys a Rapid-Equilibrium Ordered mechanism, in which phosphoenolpyruvate is the first substrate to bind and 3-deoxy-D-arabino-heptulosonate 7-phosphate is the second product to be released, rather than a Ping Pong mechanism as has been reported previously. The inhibition by tryptophan was found to be parabolic competitive with respect to D-erythrose 4-phosphate and parabolic non-competitive with respect to phosphoenolpyruvate. The enzyme was inactivated by EDTA, and could be protected against this inactivation by phosphoenolpyruvate or 3-deoxy-D-arabino-heptulosonate 7-phosphate but not by D-erythrose 4-phosphate, tryptophan or Pi. This suggests that the enzyme may be a metalloenzyme.     

Purification and properties of tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The aroH gene of Escherichia coli, which encodes the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isoenzyme of the common aromatic biosynthetic pathway, was cloned behind the tac promoter in expression plasmid pKK223-3. The enzyme was overexpressed, purified to homogeneity, and characterized. The native enzyme was found to be a dimeric metalloprotein containing 0.3 mol of iron per mol of subunit and variable amounts of zinc. The activity of the native enzyme was stimulated two- to threefold when assayed in the presence of Fe2+ ions. Pretreatment of the enzyme with Fe2+ also resulted in activation, accompanied by an equivalent increase in iron content. Treatment of the enzyme with chelating agents led to inactivation, which was fully reversed by the presence of Fe2+ in the assay mixture. The native enzyme exhibited a unique absorption profile, having a shoulder of absorbance on the aromatic band with a maximum around 350 nm and a broad, weak band with a maximum around 500 nm. Treatment of the enzyme with Fe2+ enhanced the absorbance at 350 nm and eliminated the band at 500 nm. Treatment with reducing agents caused the disappearance of both bands and destabilized the enzyme. Feedback regulation of the activity of the enzyme was specific for tryptophan, with maximum inhibition at about 70%.     

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.     

Inhibition of tyrosine-sensitive 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase by glyphosate in Candida maltosa

Abstract A gene block controlling sucrose-fermenting ability, nisin resistance and nisin production was found to be transmissible by a conjugation-like process. The 'pSN' (sucrose nisin) plasmid was transferred from 8 different nisin-producing donor strains into MG1614, a plasmid-free derivative of Streptococcus lactis 712. In the new host low yields of a plasmid of approx. 30 MDa were isolated but its authenticity as a pSN plasmid has not yet been established. Possibilities for increased nisin yield by genetic manipulation in S. lactis 712 must exist.     

New insights into the metal center of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.

Metal binding properties for a series of metal-substituted forms of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, DAHPS(Tyr), have been followed by UV-vis and EPR spectroscopy. The results show that there are two metal species present at pH = 7.0 and these are coordinated in a distorted metal binding site with a mixed nitrogen and oxygen donor atom coordination set. There is no spectroscopic evidence for strong M-S interactions in this system at any pH. Metal saturation occurs at a substoichiometric ratio of 0.8-0.85 metal/monomer, and the binding trends mirror previously published enzyme activity profiles. There is a conformational change for CuDAHPS under basic conditions, and equivalent protein handling for apoDAHPS leads to apparent loss of metal binding ability. Addition of the substrate PEP does not alter the UV-vis spectra, but there are small changes in the EPR spectra of CuDAHPS(Tyr). Further addition of the substrate analogue A5P has no effect on either spectra. Taken together, these results serve to link previous studies on enzyme activity with the recently determined X-ray crystal structure for DAHPS(Phe) and represent the first detailed spectroscopic characterization of the metal binding properties of DAHPS(Tyr).     

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.