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Research Article
1 July 2001

Microbial Origin of Plant-Type 2-Keto-3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthases, Exemplified by the Chorismate- and Tryptophan-Regulated Enzyme from Xanthomonas campestris

ABSTRACT

Enzymes performing the initial reaction of aromatic amino acid biosynthesis, 2-keto-3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthases, exist as two distinct homology classes. The three classic Escherichia coli paralogs are AroAI proteins, but many members of theBacteria possess the AroAII class of enzyme, sometimes in combination with AroAI proteins. AroAII DAHP synthases until now have been shown to be specifically dedicated to secondary metabolism (e.g., formation of ansamycin antibiotics or phenazine pigment). In contrast, here we show that the Xanthomonas campestris AroAII protein functions as the sole DAHP synthase supporting aromatic amino acid biosynthesis. X. campestris AroAII was cloned in E. coli by functional complementation, and genes corresponding to two possible translation starts were expressed. We developed a 1-day partial purification method (>99%) for the unstable protein. The recombinant AroAII protein was found to be subject to an allosteric pattern of sequential feedback inhibition in which chorismate is the prime allosteric effector.l-Tryptophan was found to be a minor feedback inhibitor. An N-terminal region of 111 amino acids may be located in the periplasm since a probable inner membrane-spanning region is predicted. Unlike chloroplast-localized AroAII of higher plants, X. campestris AroAII was not hysteretically activated by dithiols. Compared to plant AroAII proteins, differences in divalent metal activation were also observed. Phylogenetic tree analysis shows that AroAII originated within theBacteria domain, and it seems probable that higher-plant plastids acquired AroAII from a gram-negative bacterium via endosymbiosis. The X. campestris AroAII protein is suggested to exemplify a case of analog displacement whereby an ancestral aroAI species was discarded, with thearoAII replacement providing an alternative pattern of allosteric control. Three subgroups of AroAIIproteins can be recognized: a large, central group containing the plant enzymes and that from X. campestris, one defined by a three-residue deletion near the conserved KPRS motif, and one possessing a larger deletion further downstream.
Microorganisms and plants engage 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15 ) as the initial catalyst for the commitment of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) to the biosynthesis of aromatic amino acids, vitamin K, folic acid, and ubiquinone (4). The pathway contributes heavily to the biochemical individuality of organisms, with an enormous variety of peripheral branch connections known. Various pigments, siderophores, signaling compounds, defense metabolites, structural compounds, antibiotics, and other secondary metabolites are derived from this pathway (4).
DAHP synthases fall into two distinct homology families. The AroAI family is divided into subfamilies AroA and AroA (32). The AroA subfamily is widely represented in gram-negative bacteria in which differentially regulated paralogs are commonly expressed, as exemplified by Escherichia coli (three paralogs). DAHP synthase members of the AroA subfamily are more closely related to 3-deoxy-d-manno-octulosonate 8-phosphate synthases than to members of the AroA subfamily of DAHP synthases (32).
The AroAII family of DAHP synthases was first identified in higher plants in which the member proteins are chloroplast localized (11), subject to sequential feedback inhibition byl-arogenate (8), and hysteretically activated by dithiothreitol (DTT) (7, 11). The eventual availability of sequence data for these plant-type DAHP synthases revealed that they comprise a separate homology group quite distinct from the AroAI family (4). Microbial AroAIIDAHP synthases were later identified in Neurospora crassaand in several species of Streptomyces (34). Species of Pseudomonas that make phenazine pigments employ a pathway encoded by genes which include an AroAII type of DAHP synthase (21, 23). Other microbial AroAIIproteins have a specialized role in antibiotic biosynthesis (3, 5, 17, 25, 30). Thus, the emerging perspective is that microbial AroAII enzymes generally participate in a mode of secondary metabolism in which various antibiotic agents are made. In this context two general roles for AroAII can be discerned as follows. (i) AroAII is necessary for a crucial catalytic step for the production of a molecule (e.g., 3-amino, 5-hydroxybenzoate) acting as a starter unit for polyketide assembly, as exemplified by organisms producing ansamycin antibiotics (3, 5, 18) or rapamycin (25). (ii) AroAII is important for generating precursors for anthranilate synthesis. Anthranilate is then incorporated into phenazine structures (21, 23) or into menaquinone-like structures which inhibit electron transport (30). The type-i AroAII proteins apparently possess an altered substrate specificity in which either an aminated derivative of E4P is recognized or an additional overall aminating capability exists (17), whereas type-ii AroAIIproteins possess normal substrate specificity.
Most microbial AroAII proteins annotated in the National Center for Biotechnology Information's nonredundant and Finished and Unfinished Genomes databases were identified by sequence inference and by context of operon organization without any enzymological characterization, e.g., the phenazine pigment operons (21, 23). The most detailed characterization of AroAIIDAHP synthases has been from Streptomyces andNeurospora, where sequence information was incomplete at the time (34). This paper presents the enzymological properties of an AroAII DAHP synthase that exhibits primary biosynthetic function and represents a distinctive allosteric control pattern.

MATERIALS AND METHODS

Bacterial strains, plasmids, media, and growth conditions.

The Xanthomonas campestris and E. coli strains and plasmids used in this work are listed in Table1. Growth media for X. campestris and E. coli included Luria broth (LB) as a complete medium. ARO minimal medium is a modification of the medium reported by Ray et al. (24). It had the following composition (in grams per liter): glucose (1), K2HPO4 (7), KH2PO4 (2), (NH4)2SO4 (0.5), ferric ammonium citrate (0.32), NaCl (0.5), and Casamino Acids (5). After autoclaving, the following compounds (grams per liter) were added:p-aminobenzoic acid (0.01), p-hydroxybenzoic acid (0.01), 2,3-dihydroxybenzoic acid (0.01), thiamine-HCl (0.0005), and 1 ml of 1 M MgSO4. For induction of the tacpromoter, IPTG (isopropyl-β-d-thiogalactopyranoside) was added to give a final concentration of 0.2 mM. Ampicillin was used when required at a final concentration of 100 μg/ml.
Table 1.
Table 1. Strains and plasmids
Strain or plasmidRelevant characteristicsSource or reference
E. coli strains  
 XL1 Blue MRΔ(mcrA)183Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lacStratagene
 AB3248aroF363 aroG365 aroH367 proA2 argE3 elv-7 his-4 lac gal-2 tsx-358 thi35
 BL21(DE3)FompT hsdSB (rBmB) gal dcm; with DE3, a λ prophage carrying T7 RNA polymeraseNovagen
 DH5αFe14(mcrA) hsdR514 (rKmK+) recA1 endA1 gyrA96 thi-1 relA1 supE44Gibco-BRL
X. campestris strain  
 ATCC 33436 20
Plasmids  
 pTacIQColE1 ori, lacI, tacpromoter, AmprVan Kimmenade
 pTacIQ2.3Derivative of pTacIQ containing a 6.5-kb fragment of X. campestris DNAThis work
 pTacIQ2.3BDerivative of pTacIQ containing a 2.7-kb fragment of X. campestris DNAThis work
 pET-24b+Contains the T7/lacO promoterStratagene
 pET-M1Derivative of pET-24b+ containing a PCR product encoding AroAII starting from the first putative translation start siteThis work
 pET-M2Same as pET-M1 but containing a PCR product encoding AroAII starting from the second putative translation start siteThis work

Materials.

Enzymes for molecular genetic applications were purchased from New England BioLabs or Boehringer Mannheim and were used according to the specifications of the manufacturer. Chorismate purified from the accumulation medium of the triple auxotrophKlebsiella pneumoniae 62-1 (ATCC 25306) was prepared as the free acid (12) and was 97% pure. ZnSO4, MgCl2, NiCl2, and MnCl2 (puratronic grade) were obtained from Johnson Matthey (Ward Hill, Mass.); other metal salts (reagent grade) were from Sigma (St. Louis, Mo.). EDTA (analytical reagent grade) was purchased as the disodium salt from Mallinckrodt (Paris, Ky.). High-grade Spectra/Por dialysis tubing (VWR) was boiled in 2 mM EDTA and exhaustively washed before use. Chelex-treated water was used for preparation of fresh metal solutions. FeSO4 solutions were always prepared immediately before assays were performed by dissolving it in 0.01 N trace metal grade HCl (Fisher Scientific). Enzyme grade ammonium sulfate was from Sigma.

General DNA techniques and sequencing.

Cloning experiments were carried out according to the methods described by Sambrook et al. (27). The DNA sequence of aroAIIand flanking 5′ and 3′ regions was determined from plasmid pTacIQ and selected subclones and by primer walking by the dideoxy chain termination procedure (28) using a Taq FS dye terminator fluorescence-based cycle sequencing kit (Applied Biosystems) with a Perkin-Elmer/Applied Biosystems Model 377-18E1 sequencer.

Preparation and screening of a genomic DNA library.

Total genomic DNA from X. campestris was prepared following the method described by Silhavy et al. (31) and was partially digested with Sau3AI. Genomic DNA fragments from about 1 to 7 kb in length were isolated from an agarose gel and ligated toBamHI-digested pTacIQ. We had observed that direct transformation of an X. campestris genomic library into an McrA and McrB strain resulted in a lower number of transformants than in a strain without an Mcr system. For this reason, the ligation mixture was used to transform the mcrA mcrCB E. coli strain XL1 Blue MR. Plasmid DNA from the XL1 Blue MR library was purified and used to transform strain AB3248. Transformants were selected on ARO medium plates containing 100 μg of ampicillin per ml and 0.2 mM IPTG.

Strains and growth of cultures.

E. coli AB3248, a triple mutant deficient for all three DAHP synthase paralogs, was grown on LB or on minimal medium plus aromatic amino acids required for growth. E. coli DH5α and E. coli BL21(DE3) were grown on LB plus kanamycin when transformed with a plasmid. X. campestris ATCC 33436 (wild type) was grown in either LB or minimal medium supplemented with trace minerals and aromatic amino acids where appropriate.

Clones and plasmids.

Plasmid pTacIQ2.3 contains a 6.5-kb DNA segment from X. campestris. Plasmid pTacIQ2.3B contains a 2.7-kb fragment of X. campestris DNA from plasmid pTacIQ2.3, which carries the gene for DAHP synthase and complementing activities. Plasmid pTacIQ2.3B was used to prepare the pET-24b+ plasmid derivatives pET-M1 and pET-M2 carrying the M1 and M2 clones of DAHP synthase, respectively.

Preparation of clones M1 and M2.

Oligonucleotides containing an NdeI restriction site were made from the two possible 5′ ATG starts of the DAHP synthase nucleotide sequence, DAHPAI (5′-GATAAACATATGGGTCCGGCTGCGGCCGGCGTC-3′) and DAHPBI (5′-GATAAACATATGTCCGCTTCTTCCCCCTCGAC-3′). An oligonucleotide denoted DAHPSAII with an EcoRI restriction site and corresponding in reverse to the 3′ end of the sequence was also made (5′-GATAAGAATTCCTGCGCAGTCGTTTTGATC-3′). PCRs which included one each of the 5′ oligonucleotides and the 3′ oligonucleotides and the 2.8-kb fragment were performed. The PCR products were gel purified and subjected to digestion with NdeI and XhoI. The same restriction endonuclease digestion was performed on the pET-24b+ plasmid, which was then purified by gel electrophoresis. The PCR fragments were ligated with the plasmid, transformed into DH5α cells, and spread on LB-kanamycin plates. Several colonies were chosen for analysis. DNA from plasmids containing M1 (pET-M1) and M2 (pET-M2) start sites was purified, subjected to nucleotide sequencing to verify the correct sequence for each, and transformed into BL21(DE3) competent cells to obtain strains designated M1 and M2. A colony from each strain was inoculated into 10 ml of LB-kanamycin for overnight growth at 30°C. These cultures were used to inoculate 10 ml of LB-kanamycin the following morning. IPTG was added when cells reached a density of about 0.6 atA600 and were harvested about 2.5 h later. Pellets were used immediately to prepare extracts.

Extract preparation.

Cell pellets harvested from M1, M2,X. campestris, or E. coli AB3248 were suspended into 100 mM potassium phosphate buffer at pH 7.5 containing 1.0 mM DTT, 1.0 mM benzamidine, and 0.1 mM phenylmethylsulfonyl fluoride. Cell suspensions were passed at 18,000 1b/in2 through a French pressure cell, and the debris was pelleted by centrifugation at 20,000 × g for 30 min. Crude extracts to be used for enzyme assays were desalted on 10DG columns (Bio-Rad).

Enzyme assays.

DAHP synthase activities were assayed by use of the thiobarbituric acid assay as described before (15). Unless indicated otherwise, assays were carried out at 37°C in 50 mMN-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid) (EPPS) buffer at pH 8.5.

Enzyme partial purification.

For purification, a 0 to 25% ammonium sulfate fraction precipitate was centrifuged in a Sorval centrifuge for 20 min at 4°C. The supernatant fluid was further fractionated to 45% ammonium sulfate and centrifuged. The remaining supernatant was brought to 70% saturation with ammonium sulfate and centrifuged. All pellets were suspended in 50 mM EPPS buffer at pH 8.5, dialyzed for 6 h at 4°C with three buffer changes, and assayed for enzyme activities. Most of the activity was found in the 25 to 45% precipitate fraction, but a small amount was found in the 0 to 25% fraction.
Aliquots of 10 ml from the 25 to 45% ammonium sulfate fractionations were applied to a fast protein liquid chromatography (Amersham Pharmacia Biotech) MonoQ preparative column and washed with 50 mM Epps buffer as described above. A 0 to 0.5 M KCl gradient (70 ml) was applied, and 2-ml fractions were collected. A 1 M step gradient was used to remove all remaining protein. DAHP synthase activity was pooled from tubes which eluted at about the 0.24 M portion of the KCl gradient. This enzyme fraction was used in most of the assays for characterization.

Antibody preparation.

Regions from the pretransmembrane sequence (periplasmic) and from the posttransmembrane sequence (cytosolic) of X. campestris DAHP synthase were chosen for analysis to determine the best stretch of sequence to use for peptide synthesis and production of antipeptide antibodies. The chosen regions were analyzed by the use of the Network Protein Sequence Analysis at Institut de Biologie et Chimie des Protéines, Lyon, France, to predict antigenic determinants, antigenicity, chain flexibility, and hydrophobic character. After the most favorable sequences for peptide synthesis on each side of the transmembrane region were chosen, they were submitted to Aves Labs, Inc. (Tigard, Oreg.), for further analysis and synthesis. An N-terminal cysteine residue and a spacer amino acid (amino caproic acid) were added to each peptide. These additions allow the use of crude peptide for conjugation purposes and serve to avoid potential problems with steric hindrance.
The synthesized peptides were used to inoculate chickens which lay about one dozen eggs in about 14 days and can produce about 1 g of purified antibody. The highly enriched immunoglobulin fraction from the yolks was then purified (about 500 mg of immunoglobulin Y immune serum antibody). Preimmune serum for use in Western blots and electron microscopy studies was also obtained.

SDS-PAGE and Western blot analysis.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with a Mini-Protein II cell (Bio-Rad) by using the method of Laemmli (19). Crude extracts were usually diluted 1:1 (vol/vol) with solubilization buffer and heated to 100°C for 10 min. Whole-cell lysis was performed by resuspending the pellet in 2× SDS sample buffer, vortexing vigorously to shear the chromosomal DNA, and heating to 70°C for about 5 min. Samples ranging from 5 to 10 μl were loaded onto SDS–12% polyacrylamide gels. MultiMark (Novex, San Diego, Calif.) multicolored standards were used as protein markers in order to estimate the efficiency of protein transfer and to determine the molecular weight of proteins of interest.
A Trans-Blot SD semidry transfer cell (Bio-Rad) was used to transfer the protein from the mini-gels to nitrocellulose membranes sandwiched between extra-thick blot-absorbent filter paper (Bio-Rad) layers for about 15 to 30 min. Detection of antigens was performed with a chemiluminescence detection system (femtoLUCENT; GENO TECH) according to procedures recommended by the manufacturer.

RESULTS

Cloning of a DNA fragment with DAHP synthase activity.

The strategy followed to clone the gene coding for DAHP synthase fromX. campestris was to transform an E. coli strain devoid of DAHP synthase activity with a genomic library from X. campestris, followed by selection for growth in the absence of exogenous l-tryptophan. E. coli strain AB3248 lacks DAHP synthase activity due to mutations that inactivate each of the three genes coding for the differentially regulated paralog isozymes present. This strain is not able to grow in a minimal medium unless all aromatic amino acids and vitamins are added (35).
Chromosomal DNA from X. campestris was partially digested with Sau3A, ligated into the unique BamHI site present downstream of the tac promoter in the expression vector pTacIQ, and transformed into strain XL1 Blue MR. Plasmid DNA purified from this strain was used to transform strain AB3248, which was then plated on ARO minimal medium containing ampicillin (the selecting antibiotic) and IPTG (to induce the tac promoter present on plasmid pTacIQ). From about 30,000 CFU, four colonies emerged on the ARO medium plates after 48 h of incubation at 37°C. To test for reversions of any of the DAHP synthase isozyme mutant genes, plasmid DNA was isolated from each of these four transformants and used to retransform strain AB3248. Only one of the plasmids restored growth of strain AB3248 on minimal medium. This plasmid was named pTacIQ2.3. Restriction analysis of the plasmid revealed the presence of a 6.5-kb DNA segment from X. campestris. Subcloning experiments showed that a 2.7-kb partialBamHI-derived fragment present in plasmid pTacIQ2.3B was still capable of restoring the growth of strain AB3248 in ARO medium.
One possible open reading frame was deduced but had two possible translation start sites (Fig. 1). The largest (M1) was 478 amino acid residues and would have a calculated pI of 5.94 and a molecular mass of 53.4 kDa. The other (M2) would have a calculated pI of 6.14 and a molecular mass of 51.9 kDa. Both of these were subcloned into vector pET-24b+ (see Materials and Methods) to generate plasmids pET-M1 and pET-M2. These plasmids were introduced into E. coli BL21(DE3) to obtain clones M1 and M2.
Fig. 1.
Fig. 1. Sequence features of X. campestris aroAII and its gene product AroAII. (A) M1 and M2 refer to two possible translation start sites inaroAII. Putative ribosome-binding sites are highlighted in bold. (B) Schematic of X. campestrisAroAII protein features. The putative transmembrane region (residues 112 to 129) is shown in black. The location of the conserved KPRT/S motif is shown. Conserved motifs present in the putative periplasmic region and in the carboxy-terminal region are shown (x denotes nonconserved residues). X. campestris residue numbers which correspond to deletions (▵) conserved in the phylogenetic groupings indicated in Fig. 3 are shown. The large-gap cluster of organisms possesses 15 to 20 of the amino acid residues within the region corresponding to residues 188 to 243 of X. campestris, but these are not highly conserved.

DAHP synthase in crude extracts.

The specific activity of DAHP synthase (AroAII) in unfractionated extracts of wild-typeX. campestris was 5 to 8 nmol/min/mg in 50 mM potassium phosphate buffer at pH 7.0 (36). Distinctly improved specific activities (25.6 ± 1.9 nmol/min/mg) were obtained in 50 mM EPPS buffer at pH 8.3. The pH optimum was broadly similar between pHs of 8.2 and 8.5, dropping sharply under pH 8.2. Significant derepression of AroAII was not observed when a culture in the mid-exponential stage of growth in rich medium was centrifuged and resuspended in minimal medium for 2 h prior to extract preparation. As another approach, a culture in minimal medium was exposed to 10 mM glyphosate to cause starvation for aromatic amino acids (9). Although growth inhibition was observed, derepression of AroAII did not occur. Hence, the enzyme may not be subject to repression control by amino acid end products.
Crude extracts prepared from the E. coli BL21(DE3) clones M1 and M2 exhibited elevated specific activities of 796 and 3,109 nmol/min/mg, respectively. Thus, compared to the wild type, the level of AroAII in the M2 clone was enriched 25-fold or more. A preliminary series of assays comparing the enzymological properties of unfractionated extracts from the wild-type, M1, and M2 strains was undertaken to assess possible differences in catalytic properties, temperature optimum, pH optimum, and divalent cation requirements or in sensitivity to allosteric effectors. The background DAHP synthases that were present in E. coli BL21(DE3) clones were minimized by their repression to low levels in LB and the use of phenylalanine and tyrosine to inhibit the major E. coli paralogs. Characterization of the X. campestris AroAIIprotein in the E. coli AB3248 transformant was, of course, uncomplicated by any background of E. coli enzymes. We showed that purified E. coli tryptophan-sensitive DAHP available in our laboratory separated completely from X. campestris AroAII during the first step of purification. No significant differences between M1 and M2 were found. Multiple alignment results were consistent with the probability that clone M2 possesses the authentic translation start site since the length of clone M2 corresponded best with its nearest relatives, e.g., AroAII from Helicobacter pylori or fromPseudomonas aeruginosa. The initial conservation observed at the N terminus of clone M2 was 16-WSPQSW-21, which corresponded with 6-WSPTSW-11 of H. pylori and 5-WSPESW-10 of P. aeruginosa. AroAII from the M2 clone therefore seemed to be the most appropriate protein for use in detailed studies.
Crude extracts from both wild-type X. campestris and from the M1 or M2 clone in E. coli were stable at −70°C for at least 2 months. When aliquots of M2 extract that had been diluted 100-fold in 50 mM EPPS buffer (pH 8.5) were compared with aliquots diluted 100-fold in potassium phosphate buffer at pH 7.5, the latter preparations were significantly more stable in storage at −70°C. In EPPS buffer the half-life was about 4 days compared to about 24 days in phosphate buffer.

Partial purification.

Characterization of AroAIIwas carried out in EPPS buffer, the optimum buffer for catalysis. This exacerbated the problem encountered with stability, AroAIIbeing increasingly unstable with increasing purification or increasing dilution. No stabilizing conditions or agents were found, except for a modest effect of 30% glycerol. However, glycerol was not used because of interference with the chemical assay for DAHP.
In order to deal with the stability problem, a 1-day purification method was worked out for the enzymological characterization of AroAII from the M2 clone. Unfractionated extract (see Materials and Methods) was subjected to ammonium sulfate fractionation. Ninety-nine percent of the activity salted out in the 25 to 45% (NH4)2SO4 fraction. An assay after resolubilization and dialysis showed an excellent 10-fold purification (266 ± 14 nmol/min/mg). Application to an anion-exchange column yielded fractions which exhibited an additional purification of about ninefold (2.40 ± 0.16 μmol/min/mg). This protocol gave consistent preparations for characterization studies. A single major band was visualized after SDS-PAGE, but multiple minor bands were seen when the gel was overloaded. Western blots confirmed the presence of a single major band of the expected molecular weight. Further attempts to purify the enzyme from gel filtration columns were unsuccessful. Exposure of enzyme preparations, either crude X. campestrisextracts or purified protein, to antibody resulted in greater than 99% inhibition of DAHP synthase activity.

Temperature optimum.

Preliminary experiments indicated that AroAII exhibited a temperature optimum in the range of 50 to 56°C. Detailed assays were carried out in this range to locate the highest temperature at which proportionality was maintained throughout the duration of the 4-min assays used. The temperature optimum was 52°C.
The energy of activation determined according to the Arrhenius equation from a plot of log V (velocity) versus the reciprocal of the temperature (Kelvin scale) was calculated from the negative slope −E/R ( E is activation energy and R is the gas constant) and had a value of 11.9 ± 0.9 kcal mol−1.

Effect of DTT.

Since AroAII from higher plants is hysteretically activated by DTT (7, 11), the possible activation of X. campestris AroAII by DTT was examined in all strains studied and at different stages of purification. No effect of DTT was found with active preparations. Preparations that had lost some or all of the original activity were also examined, but reactivation by DTT was not found.

Effect of divalent metals.

Neither unfractionated nor partially purified extracts were affected by the presence of divalent metals, as previously noted (36). However, EDTA was a potent inhibitor and was able to completely inactivate the M2 enzyme. Inhibition by EDTA was essentially instantaneous. EDTA was not able to strip essential divalent metal moieties from X. campestrisAroAII, since dialysis of a completely inhibited preparation restored all (107%) of the original activity.
A variety of divalent metals were able to reactivate X. campestris AroAII (Table2). Similar results were obtained regardless of whether the enzyme was exposed to EDTA or the divalent metal first or whether EDTA and the test metal were added simultaneously. Co2+, Zn2+, and Ni2+ were the most effective metal additives, at 0.5 mM.
Table 2.
Table 2. Reactivation of EDTA-inactivated X. campestrisAroAII by divalent metalsa
Addition(s)% Control activity
None100
EDTA12
EDTA + MgCl217
EDTA + MnCl278
EDTA + CoCl2109
EDTA + CaCl213
EDTA + FeCl27
EDTA + ZnSO496
EDTA + CuSO47
EDTA + NiCl263
a
EDTA (0.2 mM) and a 0.5 mM concentration of the indicated metal salt were added to an assay mixture containing 13 μg of partially purified X. campestris AroAII and incubated for 4 min at 37°C.

Kinetic properties of X. campestrisAroAII.

A series of substrate saturation curves were generated at different fixed concentrations of each substrate, using elapsed reaction times of 4 min at 37°C. Conditions of proportionality were maintained with respect to reaction time and protein concentration. Replots of data obtained from double-reciprocal plots resulted in apparent Km values of 0.23 and 0.13 mM for E4P and PEP, respectively.
Analysis of inhibition data confirmed the results of Whitaker et al. (36) that chorismate competitively inhibited each substrate, whereas l-tryptophan was a noncompetitive inhibitor of each substrate. Calculated Kivalues for chorismate were 0.31 mM (for PEP) and 0.09 mM (for E4P). Calculated Ki values forl-tryptophan were 0.35 mM (for PEP) and 0.61 mM (for E4P).

Transmembrane region.

When the X. campestrisAroAII sequence was subjected to PSORT analysis (http://psort.nibb.ac.jp ), an affirmative result was obtained for a single transmembrane region that would span the inner membrane (Fig.1). Figure 2 illustrates the 18-amino-acid region predicted to span the inner membrane by the dense alignment surface method. This would place the N-terminal 111 amino acid residues in the periplasm and the C-terminal 349 residues in the cytoplasm. Similar results were obtained for most of the sequences belonging to the upper group shown in Fig.3. In contrast, members of the lower group were uniformly predicted to be cytoplasmic proteins by application of the PSORT program. The twoP. aeruginosa paralogs illustrate these AroAIIdifferences in a common organism. The biosynthetic pathway protein (upper sequence in Fig. 3) has 448 residues, of which residues 87 to 103 are predicted (certainty of 0.223) to span the inner membrane. The phenazine pathway paralog (lower sequence in Fig. 3) has 405 residues and is predicted to be cytoplasmic, with a certainty of 0.480.
Fig. 2.
Fig. 2. Putative transmembrane region of X. campestris AroAII. The graphical output shown was produced by a program which applies the dense alignment surface (DAS) method (6). A dashed horizontal line indicates the loose cutoff and a solid horizontal line refers to the strict cutoff. The sequence corresponding to the transmembrane region is shown at the bottom.
Fig. 3.
Fig. 3. Phylogenetic tree of plant-type (AroAII) DAHP synthases. A multiple alignment obtained with the Multalin program was analyzed by the neighbor-joining method using the Phylip program. The higher-plant proteins (lineage lines shown in green) are tightly clustered, form a nested set within a more divergent cluster of microbial proteins, and are most closely related to orthologs within the gram-negative bacteria. The position of theX. campestris protein is highlighted in yellow. The positions of the AroAII proteins from M. aviumand M. leprae (not shown) are almost identical to that shown for M. tuberculosis. The two Stigmatella aurantiaca AroAII proteins (Fig. 4) are not shown, but both are close homologs of P. aeruginosa_1 AroAII. The most outlying group of AroAIIproteins include those (color-coded orange) which in thePseudomonas species shown are functionally linked to phenazine biosynthesis in the stationary phase of growth. The groupings marked with dotted lines and referred to as the small-gap cluster and the large-gap cluster in Fig. 1 are defined by the conserved deletions shown. Proteins are denoted in magenta in cases where complete genomic sequencing or other evidence supports the conclusion that AroAII is the sole enzyme for primary aromatic amino acid biosynthesis. Of the two AroAII proteins fromStreptomyces coelicolor in the database, the one studied by Walker et al. (34) can be identified from their peptide sequencing and is denoted with an asterisk. The latter enzyme functions in primary amino acid biosynthesis since it was the only species detected during active growth. The P. aeruginosa_1 AroAII DAHP synthase is undoubtedly a member of the DS-TRP(cha) class of tryptophan- and chorismate-sensitive enzymes (1). Variant AroAII proteins that function as amino-DAHP synthases and that are known to function in ansamycin antibiotic production are shown in blue.

KPRS motif of AroAII.

Elucidation of the crystal structure of the phenylalanine-sensitive paralog of AroAI(29) reveals that the crucial amino acid residues which coordinate with metal, PEP, and E4P are scattered throughout the primary sequence between C61 and D326. The most compact array of crucial E. coli AroAI residues is the motif 97-KPRT-100. K97 is bridged by water to a divalent metal and also coordinates with the carboxylate moiety of PEP. R99 is inferred to be an E4P-coordinating residue. The AroAIIfamily enzymes all share a very similar motif (140-KPRS-143 in X. campestris). The nearby E. coli AroAIresidue R92, which coordinates with PEP, may be equivalent to X. campestris R133, which is completely conserved in AroAII proteins.
Another motif of interest is E. coli AroAI265-DxxHxN-270, from which H268 coordinates with both PEP and divalent metals. The X. campestris AroAIImotif 374-DxxHxN-379 may be a functional equivalent since this motif is conserved throughout the AroAII family.

DISCUSSION

Comparison of X. campestris AroAII with higher-plant-type AroAII.

The plastid-localized AroAII in higher plants (denoted DS-Mn) was the first AroAII species to be extensively characterized. All higher-plant-type AroAII proteins share a number of distinctive properties (7, 8, 22). Since the microbial and higher-plant-type AroAII proteins exhibit relatively high amino acid sequence identities, it was of interest to determine what properties of plant AroAII might also typify X. campestris AroAII. Both proteins function in primary aromatic amino acid biosynthesis, and it was shown that in each case (8, 36) a single enzyme species is tightly regulated via an allosteric pattern of control called sequential feedback inhibition (16). However, plant AroAII and X. campestris AroAII recognize different key allosteric effectors, l-arogenate and chorismate, respectively. This reflects different biochemical pathway steps diverging from prephenate in these organisms (36). The minor sensitivity of X. campestris AroAII to inhibition byl-tryptophan is paralleled by a similar sensitivity noted for plant AroAII in at least one case (mung bean) (26). AroAII from either source is quite labile and exhibits a relatively high temperature optimum. The putative transmembrane region of X. campestris AroAIIaligned with a hydrophobic region in higher plants, which may also be a transmembrane region. In contrast to the aforementioned similarities, a number of striking differences are also apparent.
Higher-plant-type AroAII exhibits dramatic hysteretic activation by DTT and is thought to be under redox control influenced by endogenous thioredoxin (11). X. campestrisAroAII was not affected by DTT, and inactive enzyme preparations could not be rejuvenated with DTT. In addition, whereas higher-plant-type AroAII displays sigmoid substrate saturation curves with respect to either E4P or PEP, X. campestris AroAII produced straightforward first-order kinetics.
Higher-plant-type AroAII has activity in the absence of added metal and is stimulated about 50% with the addition of 0.5 mM Mn2+. The enzyme is readily stripped free of metal by EDTA treatment to give an inactive preparation which can be restored to original activity levels by Ca2+. It has been suggested (8) that two metal-binding sites may exist, one binding either Ca2+ or Mn2+ and the other binding only Mn2+. In contrast, although X. campestrisAroAII is also active in the absence of added metals, further metal additions do not stimulate activity. Thus, X. campestris AroAII seems to be saturated with firmly bound metal. While EDTA is effective as an inhibitor of activity, it is not able to strip the metal from the enzyme. Mn2+ was able to displace the EDTA-metal complex from the X. campestrisAroAII, but Ca2+ was ineffective and Co2+ and Zn2+ were superior to Mn2+.
The AroAII protein is probably conserved throughout theXanthomonas genus since Xanthomonas hyacinthi(strain ICPB-XH110) was previously reported (13) to possess a DAHP synthase that was 88% inhibited by 0.07 mM chorismate and 33% inhibited by 0.07 mM l-tryptophan.

Independent evolution of tryptophan-inhibited AroA enzymes.

With respect to microbial AroA enzymes that are sensitive to feedback inhibition by l-tryptophan, three classes have been recognized by this criterion (1). First, DS-TRP (exemplified by E. coli) is exclusively subject to inhibition by l-tryptophan. Second, DS-TRP(cha) recognizesl-tryptophan as a major inhibitor and chorismate as a minor inhibitor (exemplified by P. aeruginosa). Third, DS-CHA(trp) recognizes chorismate as a major inhibitor and l-tryptophan as a minor inhibitor. It was proposed (1) that the DS-TRP(cha) gene might have arisen from a fusion between genes encoding an ancestral feedback-insensitive AroA and the large subunit of anthranilate synthase, the latter providing a single source of binding sites for both chorismate and l-tryptophan. Since anthranilate synthase is an aminase, this idea is even more intriguing for the amino-DAHP synthase class of AroAII (Fig. 3), which introduces an amino group into the reaction (17). However, entry of various AroAII sequences as queries in PSI-BLAST (National Center for Biotechnology Information) did not return hits for anthranilate synthase.
Since the tryptophan-sensitive E. coli AroAIprotein belongs to a homolog class different from that of tryptophan-sensitive members of AroAII, it had an independent origin as a paralog member of the AroAI family. It is interesting that both E. coli and N. crassapossess three differentially controlled isoenzymes of DAHP synthase (14), but whereas E. coli expresses three paralogs of AroAI, N. crassa expresses two paralogs of AroAI (phenylalanine and tyrosine regulated) and one of AroAII (tryptophan regulated). Thus, these classic three-isoenzyme systems, which exhibit a similar functional pattern, differ in that the tryptophan-regulated enzymes had independent evolutionary origins.

AroAII proteins have a microbial origin.

Figure 3shows that AroAII proteins from higher plants are highly conserved and are nested inside a larger and less conserved (more ancient) microbial grouping. The most closely related sequences of theBacteria include species of Pseudomonas, Campylobacter, Rhodobacter, and Xanthomonas. Although it has been suggested that members of the Bacteria domain might have acquired AroAII from higher plants (21), the tree shown in Fig. 3 clearly reveals an ancient origin of this AroA family within the Bacteria. Thus, acquisition of AroAII by higher plants probably occurred relatively recently via endosymbiosis from a donor in the gram-negative lineage of the Bacteria.
Figure 3 shows a clear dichotomy of AroAII sequences, although this is not necessarily a functional dichotomy. The lower cluster of AroAII proteins is engaged in phenazine pigment production (8, 23) or antibiotic production. In the latter case, Streptomyces hygroscopicus uses a derivative of DAHP for synthesis of a polyketide starter for rapamycin biosynthesis (25) and Streptomyces collinus utilizes an AroAII species which produces amino-DAHP as a precursor of 3-amino, 5-hydroxybenzoate, a starter unit for napthomycin (5). These proteins lack the putative transmembrane region, are probably not sensitive to inhibition by chorismate orl-tryptophan, and are encoded by genes belonging to operons whose expression is induced during stationary-phase metabolism. These tend to be the smallest AroAII proteins, probably because of the deletion of the majority of residues within the region corresponding to X. campestris residues 188 to 243 (Fig. 2). Given the deletion of this region in proteins presumed to lack allosteric control and the variability of this region in organisms recognizing different allosteric effectors, the region corresponding toX. campestris residues 188 to 243 may very well dictate allosteric specificity. (Although the S. collinus sequence is about 100 amino acids longer than other members of the lower cluster, these amino acids are accounted for as N- and C-terminal extensions. It may very well be that the true translational start is a GTG codon that corresponds to residue 68 of the current database entry.)
The upper cluster possesses a number of AroAII proteins which presumably catalyze the formation of amino-DAHP as a precursor of ansamycin antibiotics (3, 5). Since the AroAIIprotein designated Amycolatopsis mediterranei_1 functions as an amino-DAHP synthase, the other, designated A. mediterranei_2, could serve in primary biosynthesis. However, this is not a complete genome, and we note that the AroAI class of DAHP synthases has been reported in other species ofAmycolatopsis (2, 33).
A number of AroAII proteins in Fig. 3 are from organisms whose genomes have been sequenced. Mycobacterium tuberculosis, Campylobacter jejuni, and H. pylori all possess a single DAHP synthase of the AroAII type (Fig.4). It is likely that Corynebacterium diphtheriae (and X. campestris) also possesses a single species of DAHP synthase (i.e., AroAII). Hence, these AroAII proteins must function in primary biosynthesis. Particular enzyme reactions can be performed by enzymes which evolved independently and have been referred to as analogous enzymes (10). The frequency of analog proteins which correspond to a given function is surprisingly high (10). It seems quite possible that AroAIIof X. campestris may have originally functioned as an enzyme of secondary metabolism, reminiscent of many contemporary AroAII enzymes. Chorismate-binding ability might have been a fortuitous property of the enzyme which was subsequently exploited to provide a mechanism of sequential feedback inhibition. Either this facilitated the loss of a less efficient coexisting AroAIanalog, or prior loss of the latter created selective pressure for recruitment of the AroAII analog to primary biosynthetic function.
Fig. 4.
Fig. 4. Distribution of AroA homology types in organisms whose positions are shown on a 16S rRNA-based tree. A subtree was obtained from the Ribosomal Database site (http://www.cme.msu.edu.rdp/ ). Each bar is color coded to identify any of the three types of DAHP synthase, as keyed in the legend. The plant type, Eco type, and Bsu type correspond to AroAII, AroA, and AroA (see the introduction). Multiple bars of the same color correspond to paralogs. Fusions that have resulted in attached AroQ (chorismate mutase) domains are shown in yellow. In some cases no AroA homology type is discernible (open bars). The two Neisseria Bsu type AroA proteins lack the highly conserved motif KPRT; therefore, the coding DNA sequences can be considered pseudogenes. Listing of genera from top to bottom: Methanobacterium, Archaeoglobus, Pyrococcus (three species), Methanococcus, Aeropyrum, Aquifex, Thermotoga, Deinococcus, Clostridium (two species), Enterococcus, Streptococcus (three species), Staphylococcus, Bacillus, Streptomyces, Amycolatopsis (two species),Corynebacterium, Mycobacterium (two species),Synechocystis, Chlamydia, Porphyromonas, Chlorobium, Stigmatella, Campylobacter, Helicobacter, Caulobacter, Sinorhizobium, Thiobacillus, Xylella, Bordetella, Neisseria (two species), Xanthomonas, Legionella, Pseudomonas, Shewanella, Vibrio, Pasteurella, Haemophilus (two species), Yersinia, Klebsiella, Escherichia, and Salmonella.

Phylogenetic distribution of AroAII proteins.

If the 16S rRNA tree is accepted as a gauge of phylogenetic breadth, AroAII clearly exhibits the most narrow phylogenetic distribution of the three groups of AroA proteins (Fig. 4). Thus, it must have the most recent origin. It may have arisen in association with a specialized function for secondary metabolism. WithinMycobacterium, Mycobacterium avium possesses both AroA and AroAII, whereas bothMycobacterium tuberculosis and Mycobacterium bovis possess only AroAII. We suggest that AroA was the original enzyme serving aromatic biosynthesis in Mycobacterium and has since been replaced by AroAII. Accordingly, AroA has been lost inM. tuberculosis and in M. bovis, and it may essentially be an evolutionary remnant in M. avium.
Figure 4 shows that a number of organisms other than X. campestris rely on AroAII as the sole enzyme available for aromatic amino acid biosynthesis. Although some of these organisms are nutritionally fastidious, the presence of genes specifying complete pathways to aromatic amino acids indicates the presence of a functional pathway. In addition to M. tuberculosis (and M. bovis), these include C. jejuni, H. pylori, Sinorhizobium meliloti, and Legionella pneumophila. Since DAHP synthase is a crucial focal point of allosteric regulation (albeit with remarkable variation of control patterns), one would assume that AroAII in these organisms is subject to sequential feedback inhibition as in Xanthomonas (via chorismate) or higher plants (via l-arogenate).

ACKNOWLEDGMENTS

We thank A. Van Kimmenade (Genencor International, Palo Alto, Calif.) for kindly providing plasmid pTacIQ and Gary Xie (Los Alamos National Laboratory) for assistance with phylogenetic analysis.
G.G. was supported by a fellowship from Dirección General de Asuntos del Personal Académico de la Universidad Nacional Autónoma de México.

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Recently, Birck and Woodward (M. R. Birck and R. W. Woodward, J. Mol. Evol. 52:205–214, 2001) published a phylogenetic tree that placed two higher-plant AroAII sequences with members of both the AroA and AroA subfamilies, with the inference that AroAII and AroAbelong to a common “class.” However, any tree-building program will fail to produce a valid tree without input of a proper multiple alignment. Although a few common motifs might exist (see above), there is not currently any alignment available that would provide sufficient acceptable matches for entry into a tree-building program, particularly with the large gaps that would be necessary due to the great size difference of the AroAI and AroAII protein families.

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Published In

cover image Journal of Bacteriology
Journal of Bacteriology
Volume 183Number 131 July 2001
Pages: 4061 - 4070
PubMed: 11395471

History

Received: 31 October 2000
Accepted: 3 April 2001
Published online: 1 July 2001

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Authors

Guillermo Gosset
Departamento de Microbiologı́a Molecular, Instituto de Biotecnologı́a, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62250, Mexico1;
Carol A. Bonner
Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 326112;
Roy A. Jensen
Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 326112;
Department of Chemistry, City College of New York, New York, New York 100313; and
BioScience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 875444

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