Magnetic resonance and kinetic studies of the partial complex and Component I subunit forms of Salmonella typhimurium anthranilate synthase

Metal ion interactions of the monofunctional partial complex of Salmonella typhimurium anthranilate synthase were investigated using kinetic, NMR, and EPR methods. Mn2+ activates AS-partial complex in place of Mg2+, with a Km of 0.08 microM for Mn2+ and of 3.5 microM for Mg2+ in glutamine-dependent anthranilate synthase activity. The kinetics indicated that the metal interacts at the active site with chorismate, not glutamine. EPR and NMR water proton relaxation rate (PRR) studies supported this conclusion. EPR binding analysis showed that chorismate dramatically tightens Mn2+ binding by the partial complex. PRR experiments indicated that stoichiometric amounts of chorismate cause a substantial decrease in the enhancement of water relaxation by Mn2+, while millimolar amounts of glutamine have no effect. Analysis of the frequency dependence of water proton relaxation rates yielded dipolar correlation times of 2.5 x 10(-9) s and 4.1 x 10(-9) s for the Mn2+-partial complex and Mn2+-partial complex-chorismate complexes, respectively. These studies also indicated that chorismate binding reduces the number of fast-exchanging water molecules on enzyme-bound Mn2+ from 1 to 0.25. PRR experiments with the native bifunctional anthranilate synthase-phosphoribosyltransferase enzyme indicated the existence of additional Mn2+-binding sites which presumably function to activate the phosphoribosyltransferase activity of the Component II subunit.     

Anthranilate synthase of Neurospora crassa: reaction and labeling with glutamine analogs

The multifunctional enzyme complex, anthranilate synthase from Neurospora crassa, irreversibly loses its glutamine-dependent anthranilate synthase activity on exposure to the reactive glutamine analogs DON and azaserine. Inactivation depends on the presence of the substrate chorismate, is enhanced by the cofactor Mg⁺², and is antagonized by glutamine. Inactivation correlates well with the incorporation of [¹⁴C]DON into the protein with modification localized to the β subunit (M_r 84,000) of the complex, demonstrating directly that the β subunit provides the glutamine binding site for the glutamine-dependent anthranilate synthase reaction. The slower and less extensive loss of ammonia-dependent anthranilate synthase activity indicates that maximum expression of the ammonia-dependent anthranilate synthase activity by the α subunit also depends on the interaction with an active glutamine amidotransferase domain of the β subunit.     

Chorismate aminations: partial purification of Escherichia coli PABA synthase and mechanistic comparison with anthranilate synthase

Chorismate is converted by regiospecific amination/aromatization sequences to o-aminobenzoate and p-aminobenzoate (PABA) by anthranilate synthase (AS) and PABA synthase (PABS), respectively. We report here the first partial purification of the large subunit of Escherichia coli PABA synthase, previously reported to be quantitatively inactivated in purification attempts. The subunit encoded by the pabB gene was overexpressed from a T7 promoter and purified 9-fold to 25-30% homogeneity. The pabB subunit appears unusually sensitive to inactivation by glycerol so this cosolvent is contraindicated. The Km for chorismate is 42 microM in the ammonia-dependent conversion to PABA, and we estimate a turnover number of 2.6 min-1. A variety of chorismate analogues have been prepared and examined. Of these compounds, cycloheptadienyl analogue 11 has been found to be the most potent inhibitor of Serratia marcescens anthranilate synthase (Ki = 30 microM for an RS mixture) and of the E. coli pabB subunit of PABA synthase (Ki = 226 microM). Modifications in the substituents at C-3 [enolpyruyl ether, (R)- or (S)-lactyl ether, glycolyl ether] or C-4 (O-methyl) of chorismate lead to alternate substrates. The Vmax values for (R)- and (S)-lactyl ethers are down 10-20-fold for each enzyme, and V/K analyses show the (S)-lactyl chorismate analogue to be preferred by 12/1 over (R)-lactyl for anthranilate synthase while a 3/1 preference was observed for (R)-/(S)-lactyl analogues by PABA synthase. The glycolyl ether analogue of chorismate shows 15% Vmax vs. chorismate for anthranilate synthase but is actually a faster substrate (140%) than chorismate with PABA synthase, suggesting the elimination/aromatization step from an aminocyclohexadienyl species may be rate limiting with AS but not with PABS. Indeed, studies with (R)-lactyl analogue 14 and anthranilate synthase led to accumulation of an intermediate, isolable by high-performance liquid chromatography and characterized by NMR and UV-visible spectroscopy as 6-amino-5-[(1-carboxyethyl)oxy]-1,3-cyclohexadiene-1-carboxylic acid (17). This is the anticipated intermediate predicted by our previous work with conversion of synthetic trans-6-amino-5-[(1-carboxyethenyl)oxy]-1,3-cyclohexadiene-1-carbo xylic acid (2) to anthranilate by the enzyme. Compound 17 is quantitatively converted to anthranilate on reincubation with enzyme, but at a 1.3-10-fold lower Vmax than starting lactyl substrate 14 under the conditions investigated; the basis for this kinetic variation is not yet determined.     

Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli.

The metabolic fate of p-aminobenzoic acid (PABA) in Escherichia coli is its incorporation into the vitamin folic acid. PABA is derived from the aromatic branch point precursor chorismate in two steps. Aminodeoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate and is composed of two subunits, PabA and PabB. ADC lyase removes pyruvate from ADC, aromatizes the ring, and generates PABA. While there is much interest in the mechanism of chorismate aminations, there has been little work done on the ADC synthase reaction. We report that PabA requires a preincubation with dithiothreitol for maximal activity as measured by its ability to support the glutamine-dependent amination of chorismate by PabB. PabB glutamine enhances the protective effect of PabA. Incubation with fresh dithiothreitol reverses the inactivation of PabB. We conclude that both PabA and PabB have cysteine residues which are essential for catalytic function and/or for subunit interaction. Using conditions established for maximal activity of the proteins, we measured the Km values for the glutamine-dependent and ammonia-dependent aminations of chorismate, catalyzed by PabB alone and by the ADC synthase complex. Kinetic studies with substrates and the inhibitor 6-diazo-5-oxo-L-norleucine were consistent with an ordered bi-bi mechanism in which chorismate binds first. No inhibition of ADC synthase activity was observed when p-aminobenzoate, sulfanilamide, sulfathiazole, and several compounds requiring folate for their biosynthesis were used.     

Functional expression of Arabidopsis thaliana anthranilate synthase subunit I in Escherichia coli.

Anthranilate synthase is involved in tryptophan (Trp) biosynthesis. Functional expression of subunit I from Arabidopsis (ASA1) was achieved in bacteria as a protein fused with glutathione S-transferase (GST). The active product was purified in a single step on a glutathione-Sepharose column. The Vmax (45 nmol min-1mg-1), the apparent K(M) for chorismate (180 microM), and the feedback inhibition by Trp (complete inhibition by 10 microM Trp) of the purified fusion product (GST-ASA1) were comparable to anthranilate synthase purified from plants. Polyclonal antibodies raised against the fusion project and purified by affinity chromatography on a GST-ASA1-Sepharose column cross-reacted with a 61.5-kD protein in a partially purified anthranilate synthase preparation from corn seedlings. GST-ASA1 cleavage by thrombin, as well as site-directed mutagenesis modifications of the Trp allosteric site, inactivated the recombinant protein.     

The anthranilate aggregate of Escherichia coli: kinetics of inhibition by tryptophan of phosphoribosyltransferase

The kinetic mechanism of the phosphoribosyltransferase reaction is shown to be rapid equilibrium random bi bi with an enzyme-anthranilate-pyrophosphate abortive complex. We present a rate equation that not only predicts the observed kinetic patterns but also accommodates the fact that feedback inhibition is partial, even though tryptophan (Ki = 0.5 microM) and phosphoribosylpyrophosphate (Km = 50 microM) are competitive. Neither ligand completely abolishes the effect of the other. Instead, the binding of one ligand leads to a mutual elevation in the dissociation constant of the opposing ligand by a factor of two to three. Tryptophan inhibition is noncompetitive with respect to anthranilate (Km = 0.58 microM) and does not diminish the rate of interconversion of ternary complexes. Tryptophan cooperativity, with respect to the inhibition of phosphoribosyltransferase, conforms to the concerted Monod-Wyman-Changeux formulation (kinetic Hill coefficient = 2), whereas tryptophan as an inhibitor of anthranilate synthase more closely conforms to a Koshland model of sequential cooperativity with a kinetic Hill coefficient of 1.4. The aggregate contains only one class of tryptophan sites. Thus the first tryptophan molecule bound to the aggregate maximally inhibits both phosphoribosyltransferase active centers and one of the two anthranilate synthase catalytic sites. The remaining anthranilate synthase subunit thereupon is converted into a form with less (but not zero) affinity for chorismate and a greater affinity for a second molecule of tryptophan.     

Anthranilate synthase/anthranilate 5-phosphoribosyl 1-pyrophosphate phosphoribosyltransferase from Aerobacter aerogenes

1. Anthranilate synthase and phosphoribosyltransferase from Aerobacter aerogenes purify simultaneously and sediment together on sucrose gradients, showing that they occur as an enzyme aggregate. Both activities of the intact aggregate are subject to inhibition by tryptophan. 2. By using appropriate auxotrophic mutants it was shown that an intact active enzyme aggregate is formed when the components come from separate mutant strains. An intact active aggregate can also be formed when one component is from Escherichia coli and the other from A. aerogenes. 3. Phosphoribosyltransferase of A. aerogenes is active when not in an aggregate with anthranilate synthase, but is not subject to tryptophan inhibition, indicating that the inhibitor site is on the anthranilate synthase component. 4. Anthranilate synthase can be active and sensitive to tryptophan inhibition when complexed with an inactive phosphoribosyltransferase. 5. Kinetic studies on the anthranilate synthase activity show that tryptophan is a competitive inhibitor with respect to chorismate and a non-competitive inhibitor with respect to either glutamine or NH(4) (+) ions. This is consistent with a sequential mechanism of the ordered type in which chorismate is the first reactant.     

Positive feedback effect of benzodiazepine alkaloids on enzymes of the aromatic pathway

Abstract Penicillium cyclopium produces benzodiazepine alkaloids from l -phenylalanine and anthranilate. The biosynthesis of both precursors involves the enzymes of the shikimate pathway DAHP synthase, chorismate mutase and anthranilate synthase, the latter two competing for the common substrate chorismate. After the cultures reached the phase of alkaloid production, the in vitro measurable activities of these three enzymes could be increased by adding the alkaloids during incubation. The stimulation is most pronounced with anthranilate synthase, whose activity most probably limits the rate of alkaloid formation. It is not seen with tryptophan synthase which is not involved in the formation of alkaloid precursors. The data suggest a far reaching feedback activation, coordinating precursor biosynthesis with the formation of secondary product.     

Modification of Serratia marcescens anthranilate synthase with pyridoxal 5′-phosphate

The glutamine-dependent activity of Serratia marcescens anthranilate synthase was inactivated by pyridoxal 5′-phosphate and sodium cyanide. The reaction was specific in that the ammonia-dependent activity of the enzyme was unaffected. The inactivation was stable to dilution or dialysis but was reversed by dithiothreitol. The enzyme contains dissimilar subunits designated anthranilate synthase components I (AS I) and II (AS II). Incorporation of [¹⁴C]NaCN demonstrates that modification was limited to one to two residues per AS I · AS II protomer. An active site cysteine is involved in the glutamine-dependent activity. Modification by pyridoxal 5′-phosphate and NaCN blocked affinity labeling of the active site cysteine by the glutamine analog 6-diazo-5-oxo-l-norleucine and reduced alkylation of the active site cysteine by iodoacetamide. These results suggest modification is at the glutamine active site. Initial modification by iodoacetamide did not prevent pyridoxal 5′-phosphate-dependent incorporation of ¹⁴CN showing that the pyridoxal 5′-phosphate modification did not involve the essential cysteinyl residue. These results suggest that modification of a lysyl residue in the glutamine active site of anthranilate synthase reduces the reactivity of the essential cysteinyl residue resulting in the loss of the amidotransferase activity.     

Elicitor induced secondary metabolism in Ruta graveolens L.

In vitro cultures of Ruta graveolens L. respond with rapid accumulation of acridone epoxides, furoquinolines and furanocoumarins, when challenged with autoclaved homogenate of the yeast Rhodotorula rubra. A transient increase of several enzymes of the respective biosynthetic pathways was measured but we still look for the key regulatory enzymes. We investigated whether the branch point enzymes of the shikimic acid pathway anthranilate synthase (AS) and chorismate mutase (CM) possibly play such a role. The two enzymes compete for chorismate. AS forms anthranilate, the precursor amino acid of acridone and furoquinoline alkaloids. CM channels chorismate into phenylalanine, tyrosine and phenylpropanoid biosynthesis. Elicitation resulted in a transient increase of the activity of both enzymes. Relative induction rates were 2–4 fold for AS and about 1.5 fold for CM. Constitutive CM activity, however, is about 1000 fold higher than AS activity. As in other plants 2 isoforms of CM are expected to be present in R. graveolens. A differential determination of the activity of the isoforms via the tryptophan activation rate proved to be ambiguous. Some evidence for the specific induction of a plastidic form of CM was obtained by inhibition of translation. The time courses of CM induction show CM not to be a key enzyme in elicitor induction of furanocoumarin accumulation. In comparison to other enzyme activities induction of anthranilate synthase activity corresponds closest to inducible acridone epoxide accumulation indicating a key role in its regulation. Induction of AS and CM was inhibited by actinomycin D and chloramphenicol while cycloheximid inhibited AS induction only.Abbreviations ACT actinomycin D - AS anthranilate synthase - CAP chloramphenicol - CHX cycloheximid - 4-CL 4-coumarate CoA ligase - CM chorismate mutase - DTT dithiothreitol - NMT S-adenosyl-L-methionine:anthranilic acid N-methyltransferase - PAL phenylalanine ammonia lyase - XOMT S-adenosylmethionine: xanthotoxol-O-methyltransferase     

Thermodynamics of reactions catalyzed by anthranilate synthase

Microcalorimetry and high performance liquid chromatography have been used to conduct a thermodynamic investigation of reactions catalyzed by anthranilate synthase, the enzyme located at the first step in the biosynthetic pathway leading from chorismate to tryptophan. One of the overall biochemical reactions catalyzed by anthranilate synthase is: chorismate(aq) + ammonia(aq) = anthranilate(aq) + pyruvate(aq) + H2O(l). This reaction can be divided into two partial reactions involving the intermediate 2-amino-4-deoxyisochorismate (ADIC): chorismate(aq) + ammonia(aq) = ADIC(aq) + H2O(l) and ADIC(aq) = anthranilate(aq) + pyruvate(aq). The native anthranilate synthase and a mutant form of it that is deficient in ADIC lyase activity but has ADIC synthase activity were used to study the overall ammonia-dependent reaction and the first of the above two partial reactions, respectively. Microcalorimetric measurements were performed on the overall reaction at a temperature of 298.15 K and pH 7.79. Equilibrium measurements were performed on the first partial (ADIC synthase) reaction at temperatures ranging from 288.15 to 302.65 K, and at pH values from 7.76 to 8.08. The results of the equilibrium and calorimetric measurements were analyzed in terms of a chemical equilibrium model that accounts for the multiplicity of ionic states of the reactants and products. These calculations gave thermodynamic quantities at the temperature 298.15 K and an ionic strength of zero for chemical reference reactions involving specific ionic forms. For the reaction: chorismate2-(aq) + NH4+(aq) = anthranilate-(aq) + pyruvate-(aq) + H+(aq) + H2O(l), delta rHmo = -(116.3 +/- 5.4) kJ mol-1. For the reaction: chorismate2-(aq) + NH4+(aq) = ADIC-(aq) + H2O(l), K = (20.3 +/- 4.5) and delta rHmo = (7.5 +/- 0.6) kJ mol-1. Thermodynamic cycle calculations were used to calculate thermodynamic quantities for three additional reactions that are pertinent to this branch point of the chorismate pathway. The quantities obtained in this study permit the calculation of the position of equilibrium of these reactions as a function of temperature, pH, and ionic strength. Values of the apparent equilibrium constants and the standard transformed Gibbs energy changes delta rG'mo under approximately physiological conditions are given.     

Inhibition of E. coli CTP synthase by the "positive" allosteric effector GTP

Cytidine 5'-triphosphate (CTP) synthase catalyzes the ATP-dependent formation of CTP from UTP using either ammonia or l-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as a positive allosteric effector to promote catalysis of glutamine hydrolysis. We show that at concentrations exceeding approximately 0.15 mM, GTP actually behaves as a negative allosteric effector of E. coli CTP synthase, inhibiting glutamine-dependent CTP formation. In addition, GTP inhibits NH(3)-dependent CTP formation in a concentration-dependent manner. However, GTP does not inhibit the enzyme's intrinsic glutaminase activity. Although the activation of CTP synthase by GTP does not display cooperative behavior, inhibition of both CTP synthase-catalyzed ammonia- and glutamine-dependent CTP synthesis by GTP do exhibit positive cooperativity. These results suggest that GTP binding affects CTP synthase catalysis in two ways: it activates enzyme-catalyzed glutamine hydrolysis and it inhibits the utilization of NH(3) as a substrate by the synthase domain.     

New approach to tryptophan production by Escherichia coli: genetic manipulation of composite plasmids in vitro.

For the purpose of studying the production of L-tryptophan by Escherichia coli, the deletion mutants of the trp operon (trpAE1) were transformed with mutant plasmids carrying the trp operon whose anthranilate synthase and phosphoribosyl anthranilate transferase (anthranilate aggregate), respectively, had been desensitized to tryptophan inhibition. In addition to release of the anthranilate aggregate from the feedback inhibition required for plasmids such as pSC101 trp.I15, the properties of trp repression (trpR) and tryptophanase deficiency (tnaA) were both indispensable for host strains such as strain Tna (trpAE1 trpR tnaA). The gene dosage effects on tryptophan synthase activities and on production of tryptophan were assessed. A moderate plasmid copy number, approximately five per chromosome, was optimal for tryptophan production. Similarly, an appropriate release of the anthranilate aggregate from feedback inhibition was also a necessary step to ward off the metabolic anomaly. If the mutant plasmid pSC101 trp-I15 was further mutagenized (pSC101 trp.I15.14) and then transferred to Tna cells, an effective enhancement of tryptophan production was achieved. Although further improvement of the host-plasmid system is needed before commercial production of tryptophan can be realized by this means, a promising step toward this goal has been established.     

Isolation of cyanobacterial auxotrophs by direct selection of regulatory-mutant phenotypes

We have isolated a chorismate mutase bradytroph (leaky auxotroph) ofAnabaena sp. PCC 7119 (ATCC 29151) as a spontaneous 6-fluorotryptophan-resistant mutant. The decreased chorismate mutase activity resulted in the production of quantities of the phenylalanine and tyrosine that limited rate of growth. 3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase activity in the mutant was elevated more than twofold over the wild-type activity, suggesting derepression of this enzyme. The physiological deregulation of DAHP synthase and the genetic-based deficiency of chorismate mutase promoted an elevated level of intracellular chorismate, which then overwhelmed the competitive inhibition of anthranilate synthase by tryptophan, resulting in the overproduction of tryptophan and indoleglycerolphosphate. The presence of exogenous serine increased the production of tryptophan at the expense of indoleglycerolphosphate. This indicated that the endogenous potential for increasing the amount of serine available for increased tryptophan production is limited.     

Purification and characterization of the trifunctional beta-subunit of anthranilate synthase from Neurospora crassa

The trifunctional beta-subunit of anthranilate synthase complex of Neurospora crassa has been purified from a mutant which produces no detectable alpha-subunit. The isolated beta-subunit appeared to be a highly asymmetric dimer with a s20,w of 7.35 and an apparent molecular weight of 200,000 as determined by gel filtration on Sephacryl S-300 compared with a monomer molecular weight of approximately 84,000 Da as determined by sodium dodecyl sulfate-gel electrophoresis. The purified subunit was cleaved by elastase, trypsin, or chymotrypsin into fragments which retained the three enzyme activities. After elastase digestion, two active fragments were separated by gel filtration and ion exchange chromatography. A 30,000-Da fragment, which behaved as a monomer on gel filtration, interacted with free alpha-subunit to produce glutamine-dependent anthranilate synthase activity. A second 56,000-Da fragment, which behaved as an asymmetric dimer (apparent molecular weight 140,000) on gel filtration, retained both N-(5'-phosphoribosyl)anthranilate isomerase and indole-3-glycerol phosphate synthase activity. The failure to detect an NH2-terminal amino acid residue on either the intact beta-subunit or the 30,000-Da complementing fragment, while the 56,000-Da fragment possessed an NH2-terminal histidine residue, indicated that the complementing fragment was derived from the NH2-terminal sequence of the beta-subunit.     

Study of anthranilate synthase function by replacement of cysteine 84 using site-directed mutagenesis

Cysteine 84 was replaced by glycine in Serratia marcescens anthranilate synthase Component II using site-directed mutagenesis of cloned trpG. This replacement abolished the glutamine-dependent anthranilate synthase activity but not the NH3-dependent activity of the enzyme. The mutation provides further evidence for the role of active site cysteine 84 in the glutamine amide transfer function of anthranilate synthase Component II. By the criteria of circular dichroism, proteolytic inactivation, and feedback inhibition the mutant and wild type enzymes were structurally similar. The NH3-dependent anthranilate synthase activity of the mutant enzyme supported tryptophan synthesis in media containing a high concentration of ammonium ion.     

Shikimate pathway activity in shake and fermenter cultures of Cinchona succirubra

Cell suspension cultures of Cinchona succirubra were cultivated in shake cultures and for the first time in airlift fermenters. Under both conditions L-tryptophan exerts a stimulatory effect on alkaloid formation. In this context the regulatory pattern of some shikimate pathway enzymes was investigated in non-supplemented and tryptophan supplemented Cinchona cell cultures. A remarkable increase of tryptophan decarboxylase (TDC) activity was observed in Cinchona cells under the influence of tryptophan. Apparently, like in some other indole alkaloid producing cell cultures, a high TDC activity is a prerequisite for alkaloid formation. Growth pattern and some enzyme activities of C. succirubra fermenter cultures at controlled and non-regulated pH levels were followed. Optimum growth and alkaloid formation were recorded under non-regulated (normal) pH conditions.Abbreviations TDC tryptophan decarboxylase - try L-tyrosine - phe L-phenylalanine - DAHP 3-deoxy-D-arabino-heptulosonic acid-7-phosphate - trp L-tryptophan - E-4-P erythrose-4-phosphate - PEP phosphoenolpyruvate - MDH malate dehydrogenase - G-6-PDH glucose-6-phosphate dehydrogenase - 6-PG-DH 6-phosphogluconate dehydrogenase - Ch-mutase chorismate mutase - AS-synthase anthranilate synthase - n.d. not determined     

Anthranilate synthase subunit organization in Chromobacterium violaceum

Tryptophan is an aromatic amino acid used for protein synthesis and cellular growth. Chromobacterium violaceum ATCC 12472 uses two tryptophan molecules to synthesize violacein, a secondary metabolite of pharmacological interest. The genome analysis of this bacterium revealed that the genes trpA-F and pabA-B encode the enzymes of the tryptophan pathway in which the first reaction is the conversion of chorismate to anthranilate by anthranilate synthase (AS), an enzyme complex. In the present study, the organization and structure of AS protein subunits from C. violaceum were analyzed using bioinformatics tools available on the Web. We showed by calculating molecular masses that AS in C. violaceum is composed of alpha (TrpE) and beta (PabA) subunits. This is in agreement with values determined experimentally. Catalytic and regulatory sites of the AS subunits were identified. The TrpE and PabA subunits contribute to the catalytic site while the TrpE subunit is involved in the allosteric site. Protein models for the TrpE and PabA subunits were built by restraint-based homology modeling using AS enzyme, chains A and B, from Salmonella typhimurium (PDB ID 1I1Q).     

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.