[39] Dehydroquinate synthase from escherichia coli, and its substrate 3-deoxy-d -arabino-heptulosonic acid 7-phosphate

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OF THE AROMATIC

AMINO ACIDS

[39]

phosphate, pH 7.8, 1 mM 2-mercaptoethanol, 1 mM EDTA for 1 hr. The capacity for reactivation was gradually lost after storage for 2-3 months. The course of purification is shown in Table II. The final/3 subunit preparation was homogeneous by SDS-acrylamide gel electrophoresis and amino acid sequence determination. 7'~° The molecular weight determined by SDS-acrylamide gel electrophoresis was 21,800. That calculated from the amino acid sequence 1° was 21,684. Applicability of the Methods Although these procedures will probably work well with enzymes from the pseudomonads in Group I of the Palleroni et al. u subdivision of the genus, including P. fluorescens and P. aeruginosa, they are not likely to succeed with Group II (P. cepacia, formerly P. multivorans, and relatives) and Group III (P. acidovorans and P. testosteroni) where the complex is an a2f12 tetramer and does not dissociate as easily. 5 Preliminary experiments from this laboratory have shown that the P. aeruginosa enzyme does mimic the P. putida enzyme in several column chromatographic procedures, despite considerable differences in the amino acid sequence of its fl subunit (this volume [37]). I°M. Kawamura, P. S. Keim, Y. Goto, H. Zalkin, and R. L. Heinrikson, J. Biol. Chern. 253, 4659 (1978). H N . J . Palleroni, R. Kunisawa, R. Contopoulou, and M. Doudoroff, Int. J. Syst. Bacteriol. 23, 333 (1973).

[39] D e h y d r o q u i n a t e S y n t h a s e f r o m Escherichia coli, a n d Its S u b s t r a t e 3-Deoxy-D-arabino-heptulosonic A c i d 7 - P h o s p h a t e

By SHUJAATH MEHDI, JOHN W. FROST, and JEREMY R. KNOWLES 3-Deoxy-D-arabino-heptulosonic acid 7-phosphate dehydroquinic acid + orthophosphate

Introduction Dehydroquinate synthase (DHQ synthase), the second enzyme of the shikimic acid pathway, ~ catalyzes the ring-closure of 3-deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP) to form the saturated six-meml E. Haslam, "The Shikimate Pathway," Wiley, New York, 1974.

METHODS IN ENZYMOLOGY, VOL. 142

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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DEHYDROQUINATE SYNTHASEFROME. coli CO0-

.coo-

dehydroquinate

(~)0 HO °" -""Y"I "~I0H

~)H DAHP

307

synthase NAD + M'H"

O ~ _

+ Pi OH

OH DHQ

FIG. 1. The reaction of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate catalyzed by dehydroquinate synthase.

bered ring (of DHQ) that is the precursor of the aryl group in the three aromatic amino acids and in numerous other primary and secondary metabolites in microorganisms and higher plants (see Fig. 1). The enzyme from Escherichia coli was first studied by Sprinson and coworkers, 2,3 who observed that the enzyme requires catalytic amounts of NAD + (i.e., stoichiometric with enzyme, not with substrate) for activity, and proposed a mechanistic pathway for the enzyme. The yield of enzyme from wild-type E. coli is low, however, and in order to obtain larger amounts of pure enzyme, we subcloned the gene for DHQ synthase (aroB) from plasmid pLC29-47 of the Carbon Clarke library of the E. coli K12 genome, behind the strong tac promoter in a plasmid carrying the fllactamase (ampicillin resistance) gene. 4 (The tac promoter is a hybrid of the trp and lac UV5 promoters, and is under the same control as lac.) Upon induction with the inducer isopropyl l-thio-fl-D-galactopyranoside, transformants of the E. coli strain RB 79! (RB W3110 laclqL8) overproduce DHQ synthase to the extent that the enzyme constitutes about 5% of the soluble protein of the cell. The enzyme isolated from this engineered strain has the same molecular weight (determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate) as the enzyme from wild-type E. coli. 4 More than 150 mg of the homogeneous enzyme can readily be isolated from 50 g of bacterial cells, in a two-column procedure described below. Several procedures for the chemical synthesis of the substrate DAHP have been reported. 5,6 This material may also be obtained by isolation 2 p. R. Srinivasan, J. Rothschild, and D. B. Sprinson, J. Biol. Chem. 238, 3176 (1963). 3 W. I. Maitra and D. B. Sprinson, J. Biol. Chem. 253, 5426 (1978). 4 j. W. Frost, J. L. Bender, J. T. Kadonaga, and J. R. Knowles, Biochemistry 23, 4470 (1984). 5 D. B. Sprinson, J. Rothschild, and M. Sprecher, J. Biol. Chem. 238, 3170 (1963); F. Trigalo, M. Level, and L. Szab6, J. Chem. Soc., Perkin Trans. 1 p. 600 (1970); K. H. Herrmann and M. D. Poling, J. Biol. Chem. 250, 6817 (1975); M. Adlersberg and D. B. Sprinson, Carbohydr. Res. 127, 9 (1984). 6 j. W. Frost and J. R. Knowles, Biochemistry 23, 4465 (1984).

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B I O S Y N T H E S OF I S THE AROMATIC AMINO ACIDS

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from a mutant E. coli strain, JB5, which has the phenotype AroBT y r R - . 6 This strain lacks DHQ synthase and the repressor protein that controls the transcription of two of the three DAHP synthases, and therefore accumulates reasonable quantities of DAHP. The isolation procedure for DAHP is described below.

Assay Methods Thiobarbituric Acid Assay The principle of this assay has been described. 7 Formylpyruvate from the oxidation of DAHP by periodate gives a pink adduct with thiobarbiturate, and this chromophore is estimated spectrophotometrically after extraction into cyclohexanone. The thiobarbiturate assay can be used to measure the rate of disappearance of DAHP in assays using crude enzyme, particularly during the early stages of purification. Reagents Reagent A: Sodium metaperiodate (0.2 M) in phosphoric acid (9.0 M) Reagent B: Sodium arsenite (0.8 M) in sodium sulfate (0.5 M) containing sulfuric acid (0.1 M) Reagent C: Thiobarbituric acid (0.6% w/v) in sodium sulfate (0.5 M) with the pH adjusted to 7 with sodium hydroxide Assay buffer: 3-(N-Morpholino)propanesulfonic acid (0.1 M, pH 7.4), NAD ÷ (0.15 mM, Sigma grade V), DAHP (0.66 mM dilithium salt), and cobalt chloride (1.0 mM) Procedure. Enzyme (0.02 to 0.05 units in -< 20/zl) is added to the assay buffer (2.0 ml, preincubated at 15°) and portions (200/zl) are removed every 15 or 20 sec for up to 2 min. Each portion is quenched immediately into trichloroacetic acid (100/zl, 10% v/v). Reagent A (0.1 ml) is added, and the mixture incubated at 37° for 5 min. Reagent B (0.5 ml) is then added, and the solution mixed vigorously (using a vortex mixer). Reagent C (3.0 ml) is then added and the mixture heated at 100° for 15 min. The pink chromophore is extracted into freshly distilled cyclohexanone (4.0 ml) and the absorbance of the cyclohexanone layer at 549 nm is measured after the sample has been centrifuged for 10 min, or passed through a plug of absorbent cotton. A calibration curve is constructed using known quantities of DAHP. (The extinction coefficient at 549 nm of the chromophore is between 4.8 × 10 4 and 5.6 x 104 M -1 cm -l.) The specific activity of the enzyme is expressed as the txmol of substrate consumed/min/mg protein 7 E. Gollub, H. Zalkin, and D. B. Sprinson, this series, Vol. 17A, p. 349.

DEHYOROQUINATE SYNTHASEFROME. coli

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309

(1 unit of enzyme activity = 1/zmol of DAHP consumed per min). Protein is estimated by Lowry's method s using bovine serum albumin as standard.

Phosphate Assay Purified enzyme can also be assayed by following the release of inorganic phosphate using the method and reagents described by Ames. 9 After addition of the enzyme sample to the assay buffer (2.0 ml) at 15° as described above, portions (100 /zl) are quenched into aqueous sodium dodecyl sulfate (200/zl, 1% w/v). Ascorbate-molybdate solution (0.7 ml, as described by Ames) is then added to each tube and the samples incubated at 45 ° for 20 min. After cooling to room temperature, the absorbance of the blue complex is measured at 820 nm. (The extinction coefficient at 820 nm of the chromophore is about 2.6 x l04 M -~ cm-~.) A calibration curve is constructed using standard solutions of potassium phosphate. One unit of enzyme activity = 1/zmol of phosphate released per min. Exogenous phosphatases can, of course, produce erroneously high activities when impure enzyme samples are used.

Coupled Enzyme Assay DHQ synthase activity can be assayed by coupling to dehydroquinate dehydratase and following the increase in absorbance at 234 nm.~6 DHQ synthase activity can also be assayed using the coupling enzymes dehydroquinate dehydratase and dehydroshikimate dehydrogenase/NADH, when the reaction is followed spectrophotometrically at 340 nm. This assay is described elsewhere in this volume. Enzyme Isolation Unless specified otherwise, all operations were carried out at 4 °. The NAD ÷ used in all buffers was from Sigma (grade AA-I).

Buffers A. 2-Glycerophosphate (7.0 mM), pH 6.6, containing cobalt(II) chloride (0.25 mM) and NAD + (0.5 raM) B. Potassium phosphate (75 mM) and 2-glycerophosphate (7.0 raM), pH 6.6, containing cobalt(II) chloride (0.25 mM) and NAD ÷ (0.5 mM) 8 0 . H. Lowry, N. J. Rosenberg, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 9 B. N. Ames, this series, Vol. 8, p. 115.

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C. Potassium phosphate (10 mM), pH 6.6, containing cobalt(II) chloride (0.25 mM) D. Potassium phosphate (25 mM), pH 6.6, containing cobalt(II) chloride (0.25 mM) and NAD ÷ (0.5 mM) E. Potassium phosphate (0.15 M), pH 6.6, containing cobalt(II) chloride (0.25 mM) and NAD + (0.5 mM)

Cell Growth and Lysis Single colonies of RB 791 (pJB14) from agar plates (LB, containing ampicillin at 100/xg/ml) were used to inoculate 18 tubes, each containing LB (10 ml) and ampicillin (100/~g/ml). The cells were grown overnight at 37° in a shaker bath and used to inoculate six 4 liter flasks each containing 1.0 liter of LB and ampicillin (100/xg/ml) (30 ml inoculum per flask). After growth for 1 hr at 37° in a rotary shaker, a solution of isopropyl 1-thio-/3D-galactopyranoside was added through a sterilizing membrane to each flask, to a concentration of 1.0 mM (0.238 g per flask). Growth was continued for a further 8 hr and the cells were then harvested by centrifugation at 8000 g for 10 min. The cells may be stored frozen at - 7 0 ° at this point. The cells (typically 40 g, wet paste) were suspended in buffer A (50 ml) and lysed by a single pass through a French pressure cell at 13,000 psi. The lysate was centrifuged at 27,000 g for 30 min and the supernatant (about 75 ml) was dialyzed overnight against three changes of buffer A.

Chromatography Hydroxylapatite (BioGel HTP from Bio-Rad; 400 ml wet volume) was suspended in a large volume of 2-glycerophosphate buffer (7.0 mM, pH 6.6) and the fines were removed. A column (5 x 20 cm) was poured, and then washed with the above buffer (1.5 liters) followed by buffer A (800 ml). The dialyzed cell lysate was applied to the column, followed by buffer A (400 ml). The protein was then eluted with a linear gradient (2.0 + 2.0 liters) of buffers A and B, and fractions of 20 ml were collected. Enzyme activity in the fractions was detected by a modification of the thiobarbiturate assay described above. A portion (25 /.d) of every fifth fraction was added to the assay buffer (150/xl) at 4 °. After 30 sec, the reaction was quenched into trichloroacetic acid, and the assay completed as described. Fractions containing enzyme activity (fractions 30 to 60) were combined and concentrated using an Amicon PM-10 membrane and an Amicon ultrafiltration cell under nitrogen pressure at 40 psi. Dyematrex Red A agarose gel (Amicon) (500 ml) was prepared by washing with 8 M aqueous urea (5 liters) at room temperature. After incubation in urea solution overnight, the material was washed with

[39]

DEHYDROQUINATE SYNTHASEFROME. coli

311

TABLE I PURIFICATION OF DHQ SYNTHASE FROM E. coli RB791 (pJB14)

Step

Total units

Specific activity (units/mg)

Crude lysate (from 39 g of cells) Hydroxylapatite Dyematrex Red A

6400

1.6

8300 5500

29 44

Purification (fold)

Yield (%)

1

100

18 27

129 86

buffer C (5 liters), cooled to 4 °, and the column repoured in degassed buffer C. The column was then washed with buffer C (5 liters). The sample from the first chromatography step was applied to the column after dialysis against buffer D. The column was washed with buffer D (500 ml) and eluted with a linear gradient (2.0 + 2.0 liters) of buffers D and E. Fractions containing enzyme activity were pooled and concentrated, and the concentrated enzyme solution was divided in small portions, frozen in liquid nitrogen, and stored at - 7 0 ° . The enzyme retains full activity for more than a year in storage and for more than 2 weeks at 4 ° after thawing. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate ~° showed only a single band on staining with Coomassie Blue. The purification is summarized in Table I. An alternative, more economical, protocol has also been used that employs a second hydroxylapatite column in place of the red dye column. The enzyme was precipitated from the concentrate after the first column with ammonium sulfate (to 192 g/liter). The precipitate was redissolved in buffer A (15 ml) and the solution was applied directly to a column of hydroxylapatite (150 ml, washed and equilibrated with buffer A). After sample application, the column was washed with buffer A (150 ml) and eluted with a linear gradient (500 + 500 ml) of buffers A and B. Fractions of 5 ml were collected and those containing enzyme activity were pooled, concentrated by ultrafiltration, and stored at - 7 0 °. The enzyme isolated by this procedure had the same specific activity as before, and the yield of enzyme was somewhat higher. The increase in specific activity of the enzyme from about 25 to 40 U/mg in the second chromatography step (using either the red dye or the second hydroxylapatite column) is not entirely due to the removal of contaminating protein since only one major protein band is seen on electrophoresis after the first column. The inx0 U. K. Laemmli, Nature (London) 227, 680 (1970).

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BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS

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crease may derive from the removal of an inhibitory decomposition product of NAD + (possibly adenosine diphosphoribose). A similar phenomenon has been observed with two other enzymes that have a catalytic requirement for NAD ÷ and to which the cofactor is tightly bound. ~1

Substrate Isolation We describe here only the isolation of DAHP from the growth medium of the E. coli auxotroph JBS. Since this strain grows poorly in minimal medium, the cells were allowed first to grow up in a rich medium, and were then transferred to a minimal medium of defined composition to facilitate the isolation of DAHP. The composition (per liter of medium) of the minimal medium for the growth of JB5 was as follows: KEHPO4 (7.0 g), KHEPO4 (3.0 g), (NH4)2SO4 (1.0 g), L-tyrosine (8 mg), L-phenylalanine (8 mg), L-tryptophan (4 mg), L-histidine, L-valine, and L-isoleucine (each at 40 mg), p-aminobenzoic acid and p-hydroxybenzoic acid (each at 4/~g), thiamin (1 mg), MgSO4 (0.12 g), and glucose (5 g). Solutions of glucose, MgSO4, and other salts were autoclaved separately. Solutions of the other constituents of the medium were added to the appropriate concentrations through a sterilizing membrane. Kanamycin (75/~g/ml) was present in all media. Each of 12 tubes containing minimal medium (5.0 ml) was inoculated with single colonies of JB5 from agar plates. After growth for 24 hr at 37° in a shaker bath, these cultures were used to inoculate two 4 liter flasks, each containing rich medium YT (1.0 liter) (30 ml inoculum per flask). After growth for l0 hr at 37°, the cells were harvested by centrifugation at 8000 g for I0 min. The supernatant was discarded, and the cells were then suspended in minimal medium (1.0 liter) and incubated at 37° for a further 47 hr. The suspension was then centrifuged at 8000 g for l0 min, the cells were discarded, and the medium (2.0 liters) was either stored frozen at - 2 0 ° or used directly for the next step. Each liter of accumulation medium was passed through a column (800 ml) of Dowex 50 (H + form) and the column was washed with water (1 liter). The combined eluants were concentrated by rotary evaporation at 35 °, the pH having been adjusted to 8.0 with 1.0 M LiOH. Ethanol (50 ml) was added to the residue, and then removed by rotary evaporation. Dry methanol (50 ml) was then added, and removed by rotary evaporation to dryness. The flask was cooled in ice and the solid yellowish residue was triturated with ice-cold methanol (100 ml). The residue was removed by II A. U. Bertland and H. M. Kalckar, Proc. Natl. Acad. Sci. U.S.A. 61, 629 (1963); N. H a s a n a n d E. W. N e s t e r , J. Biol. C h e m . 253, 4999 0975).

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313

filtration and washed with cold methanol (50 ml). The combined methanol extracts were evaporated to dryness, and the residue was dissolved in water (50 ml), and the pH of the solution was adjusted to 8.0 with LiOH. After removal of the water by rotary evaporation, the residue was triturated again with cold methanol. The residue was dissolved in water (100 ml), the pH of the solution was adjusted to 8.0 with aqueous triethylamine, and water was added until the conductivity of the solution was below 5 mS. This solution (200 to 250 ml) was applied to a column (2.5 × 20 cm) of DEAE-Sephadex (HCO3 form) and the column was washed with water (200 ml) and eluted with a linear gradient (1.0 + 1.0 liter) of water and triethylammonium bicarbonate (0.4 M). Fractions of about 8 ml were collected and portions from every fifth tube were assayed for DAHP by the thiobarbiturate assay and for inorganic phosphate by the Ames assay. DAHP eluted in fractions 115 to 140 and inorganic phosphate eluted in fractions 80 to 90. Fractions 25 to 60 contained dephosphorylated DAHP (formed during the fermentation and not in the subsequent work-up) which gives a positive test in the thiobarbiturate assay. The fractions containing DAHP were pooled and evaporated to dryness by rotary evaporation from repeated additions of isopropanol. The residue was dissolved in a small volume of water and passed through a small column (10 ml) of Dowex 50 (H ÷ form). The column was washed with water and the pH of the combined eluate, containing DAHP as the free acid, was brought to 4.5 with LiOH (0.25 M). The solution was freeze dried to yield a pale yellow glassy solid (350 to 400 mg of the dilithium salt) which was stored desiccated at - 7 0 °. DAHP isolated as described above was shown to be identical to chemically synthesized material by both IH and 13C NMR spectroscopy. 6 The purity was estimated enzymatically by measuring the total amount of inorganic phosphate liberated upon incubation of an accurately weighed sample either with DHQ synthase or with alkaline phosphatase. Both methods gave a purity of about 80%. This material, when treated with DHQ synthase, produces a product that is identical (by thin-layer chromatography and by IH NMR spectroscopy) with authentic dehydroquinate synthesized from (-)-quinic acid by the method of Haslam et al. ~2 Properties of DHQ Synthase M o l e c u l a r W e i g h t . The molecular weight of the enzyme was estimated to be 40,000 by denaturing gel electrophoresis and 44,000 by high-performance liquid chromatography using a gel permeation column? These

~2E. Haslam, R. D. Haworth, and P. F. Knowles, this series, Vol. 6, p. 498.

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results indicate that the enzyme is probably a monomer. The molecular weight calculated from the amino acid sequence (derived from the sequencing of the aroB gene by Coggins and his group) is 38,880 (362 amino acids).13 Specific Activity. The specific catalytic activity of the purified enzyme is 40 U/mg at 15° and pH 7.4 in the presence o f N A D ÷ (0.15 mM). The Km for DAHP has been reported to be between 33 and 50/,tM. T M Metal Ion Requirement. The nature of tightly bound metal ions, if any, is not known, though following earlier practice, COC12 was used in all buffers during the isolation of the enzyme. Dialysis of the enzyme against buffer containing ethylene-bis(oxyethylenenitrile)tetraacetate (EGTA) results in complete loss of enzyme activity. The addition of excess Co(II) restores complete catalytic activity, Zn(II) restores 15% of the catalytic activity, and Mg(II) does not restore any measurable activity.15 The DHQ synthase activity of the pentafunctional enzyme of Neurospora crassa depends on the presence of Zn(II), and DHQ synthase from mungo bean appears to be activated by either Co(II) or Cu(lI). 16,17 N A D ÷ Requirements.15 DHQ synthase has a requirement for NAD ÷ for catalytic activity as well as for structural stability of the protein, since the apoenzyme appears to be unstable. The enzyme contains 1 mol of tightly bound NAD÷/mol of protein. Gel filtration does not remove the cofactor, and treatment with charcoal results in denatured protein. The cofactor dissociates from the NAD ÷ enzyme complex with a half-life of about I0 hr. However, the cofactor readily dissociates in the form of NAD ÷ during catalytic turnover, with a dissociation half-life of the order of a few minutes. There does not appear to be any uniformity with regard to NAD ÷ binding among the few enzymes that have a catalytic requirement for NAD ÷. 18Thus, in some instances, the NAD ÷ is easily removed and full activity is restored upon the addition of NAD ÷; yet in other cases, the cofactor is extremely tightly bound and no exogenous NAD ÷ is required. Acknowledgment This article draws on work supported by the National Institutes of Health. ~3 G. Millar and J. R. Coggins, FEBS Lett. 200, I 1 (1986). ~4 p. Le Marechal and R. Azerad, Biochimie 58, 1145 (1976); P. Le Marechal, C. Froussios, M. Level, and R. Azerad, Biochern. Biophys. Res. Commun. 92, 1104 (1980). J5 S. Mehdi and J. R. Knowles, unpublished observations. 16 j. M. Lambert, M.R. Boocock, and J. R. Coggins, Biochem. J. 226, 817 (1985). ~7 E. Yamamoto, Phytochemistry 19, 779 (1980). 18 p. Frey, in "Coenzymes and Cofactors: Pyridine Nucleotides" (D. Dolphin, R. Poulson, and O. Avramovic, eds.). Wiley, New York (in press).