Pseudomonas aeruginosa possesses two novel regulatory isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

In Pseudomonas aeruginosa the initial enzyme of aromatic amino acid biosynthesis, 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase, has been known to be subject to feedback inhibition by a metabolite in each of the three major pathway branchlets. Thus, an apparent balanced multieffector control is mediated by L-tyrosine, by L-tryptophan, and phenylpyruvate. We have now resolved DAHP synthase into two distinctive regulatory isozymes, herein denoted DAHP synthase-tyr (Mr = 137,000) and DAHP synthase-trp (Mr = 175,000). DAHP synthase-tyr comprises greater than 90% of the total activity. L-Tyrosine was found to be a potent effector, inhibiting competitively with respect to both phosphoenolpyruvate (Ki = 23 microM) and erythrose 4-phosphate (Ki = 23 microM). Phenylpyruvate was a less effective competitive inhibitor: phosphoenolpyruvate (Ki = 2.55 mM) and erythrose 4-phosphate (Ki = 1.35 mM). DAHP synthase-trp was found to be inhibited noncompetitively by L-tryptophan with respect to phosphoenolpyruvate (Ki = 40 microM) and competitively with respect to erythrose 4-phosphate (Ki = 5 microM). Chorismate was a relatively weak competitive inhibitor: phosphoenolpyruvate (Ki = 1.35 mM) and erythrose 4-phosphate (Ki = 2.25 mM). Thus, each isozyme is strongly inhibited by an amino acid end product and weakly inhibited by an intermediary metabolite.     

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.     

A pair of regulatory isozymes for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase is conserved within group I pseudomonads

Two closely related subgroups of group I pseudomonads, which differ from one another in the overall enzymatic makeup of aromatic amino acid biosynthesis, possess in common the recently characterized major (tyrosine-sensitive) and minor (tryptophan-sensitive) isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Pseudomonas aeruginosa (17). Since these characterizations were made for strains whose phylogenetic positions have been determined by oligonucleotide cataloging, an initial perception of the evolution of aromatic pathway construction and regulation is emerging.     

Sequence homology between the tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Escherichia coli and hemerythrin from Sipunculida

The first enzyme of the common aromatic biosynthetic pathway in Escherichia coli, the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, contains iron as an integral part of the polypeptide chain, and the enzyme shows an absorption maximum around 350 nm (McCandliss, R.J., and Herrmann, K.M. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 4810-4813). These two properties are also found in hemerythrin, the oxygen carrier of certain marine invertebrates. The amino acid sequence of residues 10 to 18 of the enzyme from E. coli, His-Ile-Thr-Asp-Glu-Gln-Val-Leu-Met, is highly homologous to the sequence of residues 54 to 62 of hemerythrin from Phascolopsis gouldii, His-Phe-Leu-Asn-Glu-Gln-Val-Leu-Met. His54 and Glu58 of hemerythrin have previously been identified through x-ray and protein sequence analysis as iron ligands. We suggest that residues 10 to 18 of the E. coli enzyme represent part of the iron binding fold in this protein, and that His10 and Glu14 are iron ligands.     

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.     

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.     

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).     

Structural analysis of a 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase with an N-terminal chorismate mutase-like regulatory domain

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) catalyzes the first step in the biosynthesis of a number of aromatic metabolites. Likely because this reaction is situated at a pivotal biosynthetic gateway, several DAHPS classes distinguished by distinct mechanisms of allosteric regulation have independently evolved. One class of DAHPSs contains a regulatory domain with sequence homology to chorismate mutase-an enzyme further downstream of DAHPS that catalyzes the first committed step in tyrosine/phenylalanine biosynthesis-and is inhibited by chorismate mutase substrate (chorismate) and product (prephenate). Described in this work, structures of the Listeria monocytogenes chorismate/prephenate regulated DAHPS in complex with Mn(2+) and Mn(2+) + phosphoenolpyruvate reveal an unusual quaternary architecture: DAHPS domains assemble as a tetramer, from either side of which chorismate mutase-like (CML) regulatory domains asymmetrically emerge to form a pair of dimers. This domain organization suggests that chorismate/prephenate binding promotes a stable interaction between the discrete regulatory and catalytic domains and supports a mechanism of allosteric inhibition similar to tyrosine/phenylalanine control of a related DAHPS class. We argue that the structural similarity of chorismate mutase enzyme and CML regulatory domain provides a unique opportunity for the design of a multitarget antibacterial.     

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.     

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.     

Chorismate mutase and 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of the methylotrophic actinomycete Amycolatopsis methanolica.

Chorismate mutase (CM) and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS) are key regulatory enzymes in L-Phe and L-Tyr biosynthesis in Amycolatopsis methanolica. At least two CM proteins, CMIa and CMIb, are required for the single chorismate mutase activity in the wild type. Component CMIa (a homodimeric protein with 16-kDa subunits) was purified to homogeneity (2,717-fold) and kinetically characterized. The partially purified CMIb preparation obtained also contained the single DS (DSI) activity detectable in the wild type. The activities of CMIa and CMIb were inhibited by both L-Phe and L-Tyr. DSI activity was inhibited by L-Trp, L-Phe, and L-Tyr. A leaky L-Phe-requiring auxotroph, mutant strain GH141, grown under L-Phe limitation, possessed additional DS (DSII) and CM (CMII) activities. Synthesis of both CMII and DSII was repressed by L-Phe. An ortho-DL-fluorophenylalanine-resistant mutant of the wild type (strain oFPHE83) that had lost the sensitivity of DSII and CMII synthesis to L-Phe repression was isolated. DSII was partially purified (a 42-kDa protein); its activity was strongly inhibited by L-Tyr. CMII was purified to homogeneity (93.6 fold) and characterized as a homodimeric protein with 16-kDa subunits, completely insensitive to feedback inhibition by L-Phe and L-Tyr. The activity of CMII was activated by CMIb; the activity of CMII plus CMIb was again inhibited by L-Phe and L-Tyr. A tightly blocked L-Phe- plus L-Tyr-requiring derivative of mutant strain GH141, GH141-19, that had lost both CMIa and CMII activities was isolated.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Cloning of an aroF allele encoding a tyrosine-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.

In Escherichia coli, genes aroF+, aroG+, and aroH+ encode isoenzymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases that are feedback inhibited by tyrosine, phenylalanine, and tryptophan, respectively. A single base pair change in aroF causes a Pro-148-to-Leu-148 substitution and results in a tyrosine-insensitive enzyme.     

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.     

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.     

Analysis of the metal requirement of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The three isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli were overproduced, purified, and characterized with respect to their requirement for metal cofactor. The isolated isozymes contained 0.2-0.3 mol of iron/mol of enzyme monomer, variable amounts of zinc, and traces of copper. Enzymatic activity of the native enzymes was stimulated 3-4-fold by the addition of Fe2+ ions to the reaction mixture and was eliminated by treatment of the enzymes with EDTA. The chelated enzymes were reactivated by a variety of divalent metal ions, including Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mn2+, Ni2+, and Zn2+. The specific activities of the reactivated enzymes varied widely with the different metals as follows: Mn2+ greater than Cd2+, Fe2+ greater than Co2+ greater than Ni2+, Cu2+, Zn2+ much greater than Ca2+. Steady state kinetic analysis of the Mn2+, Fe2+, Co2+, and Zn2+ forms of the phenylalanine-sensitive isozyme (DAHPS(Phe)) revealed that metal variation significantly affected the apparent affinity for the substrate, erythrose 4-phosphate, but not for the second substrate, phosphoenolpyruvate, or for the feedback inhibitor, L-phenylalanine. The tetrameric DAHPS(Phe) exhibited positive homotropic cooperativity with respect to erythrose 4-phosphate, phophoenolpyruvate, and phenylalanine in the presence of all metals tested.