INTRODUCTION

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The enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) (EC 4.2.1.15) catalyzes the condensation of phosphoenolpyruvate (PEP) and D-erythrose-4-phosphate (E4P) to form DAHP and P_i. This reaction is the first committed step in the aromatic biosynthetic pathway in microorganisms and plants, from which tryptophan, tyrosine, phenylalanine, and various aromatic cofactors and secondary metabolites are derived (3, 6). DAHPS is an important control point for metabolic regulation, as it is the earliest pathway target for negative feedback control. In most microorganisms, there are multiple isoforms of DAHPS, which are distinguished by differences in the identities of their specific feedback effectors. In Escherichia coli, there are three DAHPS isozymes, namely, DAHPS(Phe), DAHPS(Trp), and DAHPS(Tyr), each sensitive to feedback inhibition by the respective aromatic amino acid. The three isozymes have a high degree of sequence similarity and undoubtedly have arisen by gene duplication and divergence (20). DAHPS(Tyr) and DAHPS(Trp) are homodimeric enzymes, whereas DAHPS(Phe) is a homotetramer.

The E. coli DAHPS isozymes have in common a requirement for a metal activator that can be satisfied in vitro by a range of divalent metal ions, including Mn²⁺, Fe²⁺, Cd²⁺, Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺ (27). The identity of the activating metal(s) in vivo has not been established unequivocally, but there is evidence for both Fe²⁺ (15, 19, 27) and Cu²⁺ (2). In vitro activation of the enzyme with Cu²⁺ or Fe²⁺ is accompanied by the appearance of a chromophore with peak A₃₅₀ (19, 27), suggesting a ligand-to-metal charge transfer between bound metal and an enzyme cysteine. There are two invariant cysteines in microbial DAHPS enzymes, namely, Cys⁶¹ and Cys³²⁸ [E. coli DAHPS(Phe) numbering]. Mutational analysis of these two residues in E. coli DAHPS(Phe) (28) has shown that Cys⁶¹ is essential for metal binding and catalytic activity. In contrast, Cys³²⁸ is nonessential, although conservative replacements at this position did have significant negative effects on the kinetic properties of the enzyme, suggesting that it lies near the active site.

It has long been known that the substrate PEP stabilizes DAHPS during purification and storage and protects the enzyme against heat inactivation (14, 17, 22). The second substrate, E4P, has the opposite effect on the stability of DAHPS, increasing the rate of spontaneous inactivation in vitro (15, 17). It has been assumed that this effect is indirect, resulting from the depletion of residual PEP by its enzymatic conversion to DAHP.

Here we show that the in vitro instability of DAHPS(Phe) results from the oxidation of its two invariant cysteines, catalyzed by metal cofactor bound at the metal site, and that PEP, bound at the active site, protects the residues against this attack. All evidence indicates that DAHPS(Phe) is a typical metal-catalyzed oxidation (MCO) enzyme system (24-26).

	MATERIALS AND METHODS

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Chemicals and enzymes. 1,3-Bis[tris(hydroxymethyl)-methylamino]propane (BTP), PEP (cyclohexylammonium salt), E4P (sodium salt), dithiothreitol (DTT), dimethyl suberimidate, 5,5'-dithiobis-(2-nitrobenzoate) (DTNB), bovine liver catalase, tosylamide phenylethyl chloromethyl ketone (TPCK)-treated trypsin, amino acids, vitamins, and antibiotics were from Sigma Chemical Co. (St. Louis, Mo.); high-purity CaCl₂, CoCl₂, CuSO₄, FeCl₃, FeSO₄, MgCl₂, MnCl₂, and ZnSO₄ were from Aldrich (Milwaukee, Wis.) or Mallinckrodt (Paris, Ky.).

Bacterial plasmids and strains. The host strain, E. coli CB735 [C600 (gal-aroG-nadA) aroF::Cat^r aroH::Neo^r recA1/F' lacI^qZM15 proA⁺ B⁺ Tn10(Tet^r)], carries a deletion and/or interruption of each of the three chromosomal genes encoding the three DAHPS isoforms (1). Strain CB717 is the recA⁺ version of CB735. Plasmid pTAG1 is a derivative of plasmid pTTG1 (28) in which the transcription of wild-type aroG is driven by one tac and two aroG promoters arranged in tandem. Mutant aroG plasmids pTAG12 (Cys⁶¹Ser) and pTAG6 (His⁶⁴Leu) are derivatives of pTAG1, and mutant plasmid pTTG16 (Cys³²⁸Val) is a derivative of pTTG1; all were constructed by oligonucleotide mutagenesis.

Purification of DAHPS(Phe). Wild-type DAHPS(Phe) was isolated from overproducing strain CB735/pTAG1. Mutant enzymes were isolated from strains CB735/pTAG6, CB735/pTAG12, and CB717/pTTG16. Growth of cultures, preparation of crude extracts, and purification of DAHPS(Phe) were done as previously described (27). Minor impurities in the final MonoQ preparation were then removed by using hydroxylapatite chromatography as follows. The peak fractions from the MonoQ chromatography were concentrated by ultrafiltration using a Centricell unit (Polysciences Inc., Warrington, Pa.) and loaded on a Bio-Gel HPHT hydroxylapatite column (7.8 mm by 10 cm; Bio-Rad, Rockville Centre, N.Y.) equilibrated with 100 mM sodium phosphate-0.01 mM CaCl₂ (pH 6.8). The column was developed at a flow rate of 0.5 ml/min with a 20-ml sodium phosphate gradient (0 to 1.0 M, pH 6.8). The DAHPS(Phe) peak emerged at approximately 400 mM sodium phosphate. PEP (200 µM) and EDTA (2 mM) were added immediately to the fractions containing DAHPS activity. The combined fractions were concentrated by ultrafiltration using a Centricon 10 unit (Amicon, Beverly, Mass.) and then dialyzed on ice against 20 to 50 volumes of metal-free BPP (20 mM BTP, 200 µM PEP, pH 6.8) with two or three changes. Metal-free BPP buffer was prepared by using Chelex 100 chelating resin (Bio-Rad). Before use, dialysis tubing was boiled in 2% sodium bicarbonate-1 mM EDTA-1 mM EGTA and then exhaustively washed with Chelex-treated water.

Determination of protein concentration. The protein concentration of crude and partially purified preparations of DAHPS(Phe) was determined colorimetrically by the method of Bradford (4) using the Bio-Rad protein assay reagent with bovine serum albumin as the standard. The concentration of purified preparations was determined spectrophotometrically at 280 nm by using a molar extinction coefficient (^M₂₈₀) of 40,500 for the DAHPS(Phe) monomer (27). The DAHPS(Phe) concentrations given are molar concentrations of the enzyme monomer.

Enzymatic assay. DAHPS activity was assayed spectrophotometrically (27) by measuring the rate of disappearance of PEP at 25°C. Reaction mixtures contained 20 mM BTP (pH 6.8), 150 µM PEP, 450 µM E4P, and, unless indicated otherwise, 50 µM Mn²⁺. Reactions were generally initiated by addition of 10 to 70 nM enzyme to the reaction mixture.

Determination of histidine and cysteine residues. Histidine residues of native and inactivated DAHPS(Phe) were titrated by treating the enzyme (3 to 5 µM) in 20 mM phosphate buffer, pH 7.0, with 0.2 mM diethyl pyrocarbonate (Aldrich) at 25°C (16). The increase in A₂₃₀ was monitored until a maximum was attained. The amount of N-carbethoxyhistidyl adduct formed was calculated by using an ^M₂₃₀ of 3,000.

Peptide mapping. DAHPS(Phe) samples were applied to a Sephadex G25 PD-10 column equilibrated and developed with 100 mM tris(hydroxymethyl)aminomethane-HCl (pH 8.2). The proteins (400 to 800 µg) were digested with TPCK-treated trypsin (20:1, wt/wt) for 24 h at 37°C. The trypsin was added to the reaction mixture in three equal portions at 8-h intervals. The digestion was terminated by addition of 1/50 volume of glacial acetic acid. The tryptic peptides (80 to 100 µg) were fractionated by reverse-phase high-performance liquid chromatography using a Vydac C₁₈ column (0.46 by 25 cm). The column was equilibrated with 5% acetonitrile-0.1% trifluoroacetic acid and developed with a linear 5 to 95% acetonitrile gradient containing 0.1% trifluoroacetic acid at a flow rate of 1%/min. The elution of peptides was monitored by measuring the A₂₁₄. Fractions were dried in vacuo at room temperature in a Savant Speedvac. N-terminal sequencing and mass spectrometry of peptides were performed at the University of Virginia Biomolecular Research Facility.

	RESULTS

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In vitro stability of DAHPS(Phe). ApoDAHPS(Phe) displayed significant instability during storage in buffer free of ligands and additives, losing activity steadily over time, with a half-life of about 1.2 days at 22°C (Fig. 1). However, in the presence of 1 mM PEP, the enzyme was very stable, losing little activity after 4 days under the same conditions. It was fortuitously discovered in these experiments that 1 mM EDTA was as effective as PEP in stabilizing the enzyme. Neither PEP nor EDTA, nor a combination of the two, was able to reactivate the partially decayed apoenzyme, but each did prevent further inactivation (data not shown).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   In vitro stability of DAHPS(Phe). ApoDAHPS(Phe) (~20 µM) was treated at 22°C with PEP (1 mM) and/or EDTA (1 mM) as indicated. Symbols: , no addition; , PEP; , EDTA; , PEP plus EDTA.

View larger version (27K): [in this window] [in a new window]   FIG. 2.   Analysis of subunit dissociation of DAHPS(Phe) by size exclusion FPLC. Enzyme preparations were incubated at 22°C for the indicated times and then fractionated on a Superose 12 column using 20 mM BTP-100 mM KCl (pH 6.8) at a flow rate of 0.5 ml/min. Protein was detected by measuring the A280. PEP and EDTA were at 1 mM, as indicated. (A) Apoenzyme incubated with either PEP, EDTA, or PEP plus EDTA. (B) Apoenzyme incubated without addition. The expected elution times of the various oligomeric forms of the enzyme, extrapolated from a standard curve prepared with a set of proteins whose molecular weights are known, were as follows: tetramer, 21.1 min; dimer, 23.3 min; monomer, 25.0 min. The dashed vertical line in panel B (2 days) marks a switchover point for the automated integration of the peaks.

Effects of metals on stability. In order to ascertain whether the spontaneous inactivation of apoDAHPS(Phe) was metal specific, the enzyme was treated for 18 h at 22°C with a variety of metal activators both in the presence and in the absence of PEP. It was found that 20 µM Cu²⁺ caused complete inactivation of the enzyme and destabilization of its quaternary structure. Fe²⁺ was partially effective, leading to 60% inactivation at 20 µM and 90% inactivation at 200 µM. Co²⁺, Mn²⁺, Zn²⁺, and Fe³⁺ had little or no effect, even at 10-fold higher concentrations. PEP (400 µM) afforded full protection from inactivation by 20 µM Cu²⁺ and Fe²⁺ and partial protection at 200 µM Cu²⁺. The lesser effects of Fe²⁺ were apparently due to the oxidation of Fe²⁺ to Fe³⁺ under the aerobic conditions of the experiment, since the addition of freshly prepared Fe²⁺ to the partially inactivated enzyme led to complete inactivation and dissociation. In light of this, Cu²⁺ was chosen for further characterization of the phenomenon of metal inactivation.

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Inactivation of DAHPS(Phe) by Cu2+. ApoDAHPS(Phe) (~20 µM) was treated at 22°C with the indicated concentrations of CuSO4 in the presence or absence of 500 µM PEP. Residual enzymatic activity was measured at the indicated times. Symbols: , no Cu2+ but no PEP; , PEP but no Cu2+; , 20 µM Cu2+ but no PEP; , 20 µM Cu2+ plus PEP; , 200 µM Cu2+ but no PEP; , 200 µM Cu2+ plus PEP.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Effect of catalase and anaerobiosis on inactivation of DAHPS(Phe) by Cu2+. ApoDAHPS(Phe) (13 µM) was treated with 200 µM CuSO4. Where indicated, PEP (400 µM) or bovine liver catalase (34,000 U) was added 5 min before addition of CuSO4. Anaerobiosis was achieved by continuous bubbling of N2 gas through the enzyme solution in a sealed tube. Bubbling was begun 5 min before the addition of CuSO4. Symbols: , no treatment; , PEP; , catalase; , anaerobiosis.

Identification of target residues. A variety of active-site residues have been found to be the target of oxidative attack in different MCO enzyme systems, including arginine, cysteine, histidine, lysine, and proline (25). In preliminary attempts to identify the affected residue(s) in DAHPS(Phe), it was found that the rate of spontaneous inactivation of apoDAHPS(Phe) was markedly influenced by pH, increasing as the pH decreased from 7.0 to 6.0. When first-order rate constants of the loss of enzymatic activity were plotted as a function of pH, the derived curve indicated a critical target residue whose pK was ~6.3, suggesting that the protonated form of histidine is the target for oxidation. However, when the number of histidine residues in spontaneously inactivated apoDAHPS(Phe) was assessed by diethyl pyrocarbonate modification, no significant reduction from the 11 residues present in the native enzyme was found. The apoenzyme treated with 200 µM Cu²⁺ showed a gradual loss of histidine residues with time, increasing from one residue lost after 5 h, at which time there was >90% inactivation, to three residues lost after 24 h. However, the same results were obtained when the metal treatment was carried out with the wild-type enzyme in the presence of 400 µM PEP, where there was little (<10%) loss of enzymatic activity, as well as with the Cys⁶¹Ser enzyme, which is defective in metal binding. Thus, the copper-catalyzed attack of histidine residues was nonspecific rather than targeted to a residue(s) at the active site of the enzyme.

Identification of oxidized cysteine residues. In order to identify the oxidized cysteine residues in the inactivated enzyme, peptide mapping of the native enzyme, the spontaneously inactivated enzyme and the Cu²⁺-inactivated enzyme was carried out. The peptide profiles of the native enzyme (Fig. 5A) and the spontaneously inactivated enzyme (Fig. 5C) were similar, although there were quantitative differences in a number of the peaks. In contrast, in the Cu²⁺-inactivated preparation, a major new peak was observed, eluting late in the fractionation (Fig. 5B). In experiments in which a range of Cu²⁺ concentrations (0.25-, 1.0-, and 10-fold molar ratios) was used for inactivation, the novel peptide increased in amount as the concentration of Cu²⁺ increased. In all cases, DTT reactivation of the Cu²⁺-inactivated enzyme preparations led to the disappearance of the novel peptide (data not shown). It was not possible to correlate the absence of a specific peak in the peptide profile of the native enzyme with the appearance of the novel peak, perhaps because of ^M₂₁₄ differences in the peptides.

View larger version (25K): [in this window] [in a new window]   FIG. 5.   Tryptic peptide maps of native and inactivated DAHPS(Phe). Peptide mapping was carried out as described in Materials and Methods. Panels: A, native apoDAHPS(Phe) (fully active); B, apoDAHPS(Phe) treated with 400 µM CuSO4 for 4 h at 22°C (>90% inactivation); C, apoDAHPS(Phe) incubated for 4 days at 22°C (>90% inactivation). The arrow in panel B indicates the novel peptide peak that appears upon Cu2+ treatment.

Stability of Cys³²⁸Val DAHPS(Phe). As mentioned above, the inactive Cys⁶¹Ser enzyme, which does not bind metal, was found to be resistant to metal attack, as judged by the lack of subunit dissociation upon Cu²⁺ treatment. This raised the question of whether a mutational change at Cys³²⁸ would also affect metal sensitivity. Accordingly, the stability of the Cys³²⁸Val enzyme, previously shown to have only slightly impaired catalytic properties, i.e., a 20% reduction in the catalytic constant and two- to threefold increases in K_m^PEP and K_m^E4P (28), was examined. It was found that the Cys³²⁸Val apoenzyme was completely resistant to both spontaneous and Cu²⁺-catalyzed inactivation (Fig. 6), demonstrating that metal attack of DAHPS(Phe) requires the presence of both the Cys⁶¹ thiol and the Cys³²⁸ thiol.

View larger version (18K): [in this window] [in a new window]   FIG. 6.   In vitro stability of Cys328Val mutant DAHPS(Phe). ApoDAHPS(Phe) (~20 µM) was treated at 22°C as indicated. Symbols: , , wild-type enzyme; , , Cys328Val enzyme. Open symbols, no added Cu2+ (days timescale); filled symbols, 250 µM CuSO4 (hours timescale).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The results reported here establish that the in vitro instability of DAHPS(Phe) in the absence of bound PEP is due to the oxidation of two active-site cysteinyl residues, Cys⁶¹ and Cys³²⁸, catalyzed by redox metal ions that normally activate the enzyme. The oxidation of thiol groups in proteins proceeds by a succession of single-electron transfers, giving rise to a progression of oxidation states (Fig. 7), all of which, except for the sulfonic acid, are reversible by DTT and other reducing agents (10, 13).

View larger version (10K): [in this window] [in a new window]   FIG. 7.   Succession of single-electron transfers giving rise to a progression of oxidation states of the thiol group.

The first two derivatives of the oxidative pathway, the thiyl radical and the sulfenic acid, are highly reactive, and if two such adducts exist in close enough proximity and are sterically free to interact, they will spontaneously form a disulfide linkage (12, 13). Thus, it is possible that during spontaneous decay of DAHPS(Phe) (i.e., trace amounts of metal ions), the thiyl or sulfenic acid derivatives of Cys⁶¹ and Cys³²⁸ are formed and, perhaps because of steric limitations and the protective environment of the active site, are able to persist as such. This is consistent with the crystal structure of the enzyme, where it has been found that Cys⁶¹ and Cys³²⁸ are in close proximity within the active site (C's within ~6 Å), but their two thiol groups are not favorably positioned for disulfide bond formation (23). For the thiols to attain optimal orientation, the side chain of Cys⁶¹ must undergo a net rotation of about 180° about its C-C bond. Thus, under conditions of high metal concentration, significant conformational changes must accompany the oxidation of the two cysteine thiols, permitting formation of the disulfide bridge.

Besides metal and O₂, MCO systems characteristically require either a nonenzymatic electron donor such as ascorbate, NAD(P)H, or thiol compounds or an enzymatic system for the generation of needed electrons (24, 25). The electron donor plays a dual role in the reduction of Cu²⁺ or Fe³⁺ to Cu¹⁺ or Fe²⁺ and in the production of H₂O₂ by the reduction of O₂. The H₂O₂ then disproportionates via Fenton chemistry to a hydroxyl radical (.OH), the reactive oxygen species ultimately responsible for oxidative damage to proteins, and a hydroxyl ion (OH) with the regeneration of the oxidized metal ion (5, 25). Since the DAHPS(Phe) system does not require an exogenous electron donor, the electrons required for H₂O₂ formation must be derived endogenously. A possibility that is consistent with the data is that the thiol group of Cys³²⁸ serves as the required electron donor.

Based on this assumption, the following mechanistic model for the DAHPS(Phe) MCO system is postulated:
(1) Cys³²⁸-SH + Cu²⁺ + O₂  Cys³²⁸-S. + Cu¹⁺ + H₂O₂
(2) Cu¹⁺ + H₂O₂  Cu²⁺ + .OH + OH
(3) Cys⁶¹-SH + .OH  Cys⁶¹-S. + H₂O
(4) Cys⁶¹-S. + Cys³²⁸-S.  Cys⁶¹-S-S-Cys³²⁸
The sequence is initiated by the reduction of O₂ to H₂O₂ with the concomitant oxidation of the thiol group of Cys³²⁸ to the thiyl radical, catalyzed by redox metal present at the active site (reaction 1). The reduced Cu¹⁺ ion formed in reaction 1 then catalyzes the formation of the hydroxyl radical from H₂O₂ via Fenton chemistry, regenerating Cu²⁺ (reaction 2). The hydroxyl radical, in turn, attacks Cys⁶¹, oxidizing its thiol group to the thiyl radical (reaction 3), thereby eliminating metal binding and inactivating the enzyme. In the spontaneously inactivated enzyme, the two oxidized cysteine thiols remain unbridged. However, in the Cu²⁺-inactivated enzyme, additional oxidative modifications of the enzyme occur, such as the attacks on the nonessential cysteine and histidine residues that were detected (Table 1), that lead to the conformational changes necessary for the repositioning of the Cys⁶¹ side chain and spontaneous formation of the disulfide (reaction 4). It is likely that the same or similar modifications occur in the spontaneously inactivated enzyme posttreated with Cu²⁺, since this treatment triggers disulfide formation in the already inactive enzyme. These nonspecific modifications presumably derive from the ability of the metal in solution to act as a free radical in the generation of activated oxygen derivatives (5) and thus would not be restricted to active-site residues.

The first three steps of the MCO model described above envisions a "caged" reaction at the active site of DAHPS(Phe) that involves the sequential oxidation of Cys³²⁸ and Cys⁶¹ by distinctly different mechanisms. It explains why modification of the nonessential Cys³²⁸ residue is required for inactivation of the enzyme (Fig. 6). It also explains how trace amounts of metal are able to accomplish the slow inactivation of a large excess of enzyme molecules (Fig. 1). Reactions 1 and 2 constitute a redox cycle that regenerates Cu²⁺ which, no longer coordinated by the oxidized thiol of Cys⁶¹, is free to exit the damaged active site and diffuse to another intact active site to repeat the cycle. It is postulated that PEP bound at the active site blocks initiation of the cycle by sequestering the bound metal, thereby protecting both Cys³²⁸ and Cys⁶¹ from oxidative attack. This is consistent with both crystallographic and ligand binding studies of the enzyme. The crystal structure of DAHPS(Phe) has revealed that the metal is coordinated by bound PEP at the active site (23), an interaction that increases the binding constant of both ligands by an order of magnitude (18). On the other hand, no insight can be derived from the crystal structure of the enzyme to explain how oxidation of the two active-site cysteines leads to subsequent destabilization of the quaternary structure of the enzyme.

In view of the findings that DAHPS(Trp) and DAHPS(Tyr) are also markedly unstable in vitro in the absence of PEP (19, 22), that the three isozymes share the same spectrum of metal activators (27), and that Cys⁶¹ and Cys³²⁸ are invariant in the three isozymes (20), it appears likely that the mechanism of metal-catalyzed oxidation found here in DAHPS(Phe) is operable in the other two isozymes as well. On the other hand, it is not clear whether metal-catalyzed oxidation plays a role in the metabolic regulation of the level of DAHP synthase activity in vivo, as has been shown in other MCO enzyme systems (24, 26). Such a mechanism would imply that the intracellular availability of PEP during growth modulates the susceptibility of the enzyme to metal-catalyzed attack and subsequent proteolytic turnover (8, 9, 21). That iron and/or copper are most likely the activating metals in vivo (2, 19, 27) is consistent with this possibility. However, little is known about the intracellular turnover of the DAHP synthase isozymes in E. coli. In one report, it was proposed that specific proteolytic degradation acts to control the level of DAHP synthase activity during growth (7). This was based on the observation that the activities of the coordinately expressed enzymes DAHPS(Tyr) and chorismate mutase became disproportionate when the cells entered the stationary phase of growth, apparently as a result of the inactivation of DAHPS(Tyr). Thus, it is possible that in the late stages of the growth of E. coli, the normally abundant intracellular pool of PEP (11) becomes depleted, leading to the metal-catalyzed oxidation and degradation of one or more of the DAHPS isozymes. A closer examination of individual isozyme turnover rates, PEP pool levels, metabolite compartmentalization, and metal utilization throughout the growth cycle is needed before the efficacy of this type of metabolic regulation can be established.