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[38] Anthranilate synthase from fluorescent pseudomonads
300
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
[38]
The trp genes of other tryptophan-synthesizing eukaryotes have not yet been thoroughly analyzed. The tendency for gene fusion exhibited by the fungi appears to have been carried further in Euglena gracilis, where all the enzymes of the pathway except AS are incorporated into a single, large polypeptide chain over 200,000 in molecular weight. 3j It should not be assumed that this situation holds for all photosynthetic eukaryotes, however, for both the green algae and plants seem to have all the activities of the pathway on separate molecules, and their TS has a and /3 subunits resembling the bacterial o n e s ) 2 Gene structure has not been thoroughly analyzed in these cases, however. 3~ j. C. L a r a and S. E. Mills, J. Bacteriol. 110, 1100 (1972). 32 j. C h e n and W. G. Boll, Can. J. Bot. 50, 587 (1972); R. T. N a g a n o and T. C. Moore, Arch. Biochem. Biophys. 149, 402 (1972); C. N. Hankins, M. T. Largen, and S. E. Mills, Plant Physiol. 57, 101 (1976).
[38] A n t h r a n i l a t e S y n t h a s e f r o m F l u o r e s c e n t P s e u d o m o n a d s
By IRVING P. CRAWFORD Anthranilate synthase, the first enzyme specific to tryptophan synthesis, has been intensively studied in a number of organisms, l The enzyme normally catalyzes Reaction (1). C h o r i s m a t e + L-glutamine ~ anthranilate + pyruvate + L-glutamate
(1)
In Pseudomonas putida and Pseudomonas aeruginosa the enzyme has an 0//3 subunit structure. The larger a subunit (component I in the nomenclature of Ito and Yanofsky2), can catalyze Reaction (2) independently. C h o r i s m a t e + NH3 ~ anthranilate + pyruvate •
(2)
The smaller/3 subunit (component II) contributes a glutamine binding site to the complex; it belongs to a family of glutamine amidotransferase molecules associated with the anthranilate and p-aminobenzoate synthases from a variety of bacteria and fungi) Because the gene for this small subunit is not fused to other pathway components and the enzyme does not self-aggregate to the 0/2/32 form, anthranilate synthase from fluorescent 1 H. Zaikin, Adv. Enzymol. 38, I (1973); in "Multifunctional Proteins" (H. Bisswanger and E. Schmincke-Ott, eds.), p. 123. Wiley, N e w York, 1980. 2 j. Ito and C. Y a n o f s k y , J. Biol. Chem. 241, 4112 (1966). 3 j. B. Kaplan, W. K. Merkel, a n d B. P. Nichols, J. Mol. Biol. 183, 327 (1985).
METHODS IN ENZYMOLOGY, VOL. 142
Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.
[38]
ANTHRANILATE SYNTHASE FROM PSEUDOMONADS
301
pseudomonads is the smallest and simplest form of the enzyme that has been studied intensively. ~Despite its structural simplicity and small size, however, the enzyme shows typical feedback inhibition by L-tryptophan. As in other, better studied instances 4 the a subunit has been shown to contain the tryptophan binding site) Two procedures have been devised for the purification of P. p u t i d a anthranilate synthase. Both rely on controlling the reversible dissociation of the subunits and employ fractionations first for the a/3 complex, then for the individual subunits. Although Queener et al. 6 achieved purity of both components, reporting a yield of 25% for the a and 41% for the/3 subunit, Goto et al. 7 had to revise this procedure considerably to obtain pure/3 subunit. Their procedure gave pure/3 subunit in 32% yield, with a subunit being only 60% pure but essentially free of/3 subunit. Both procedures will be described. Growth of Cells The a and/3 subunits of anthranilate synthase are derepressed coordinately when an auxotroph blocked later in the pathway is starved for tryptophan. The auxotroph used by Queener et al., 6 PpG281 trpF21, was a slightly leaky mutant blocked in the third step of the pathway, while that used by Goto et al. 7 was trpC621, blocked in the fourth step. As both mutants had the same P. p u t i d a parent, it seems unlikely that strain differences affected the purification of the enzyme. Because of variation in the assay conditions used by the two laboratories it is not certain that equivalent levels of derepression were obtained in the two studies, however. Assay Procedures Both methods employed the continuous fluorometric assay for anthranilate formation that has been described in earlier contributions in this series (Vol. XVIIA [46, 47, 48, 48a]; Vol. 113 [37]). Activity levels were high enough so that even crude extracts could be assayed directly in the fluorometer cuvette. Queener et al. used 25 mM Tris-C1, pH 7.8, 5 mM MgCI2, 5 mM L-glutamine, 0.25 mM chorismate, and 13 mM 2-mercaptoethanol for Reaction (1), substituting 0.2 M glycine, pH 8.8, for the Tris 4 H. Nagano and H. Zalkin, J. Biol. Chem. 245, 3097 (1970). 5 S. F. Queener and I. C. Gunsalus, Proc. Natl. Acad. Sci. U.S.A. 67, 1225 (1970). 6 S. F. Queener, S. F. Queener, J. R. Meeks, and 1. C. Gunsalus, J. Biol. Chem. 2,48, 151 (1973). 7 y . Goto, H. Zalkin, P. S. Keim, and R. L. Heinrikson, J. Biol. Chem. 25L 941 (1976).
302
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
[38]
buffer and 0.3 M (NH4)2 SO4 for glutamine to measure Reaction (2). Their reaction mixture was maintained at 37°. Goto et al. used 50 mM potassium phosphate (pH 7.4), 20 mM L-glutamine, l0 mM MgC12, 0.1 mM chorismate, and 25 mM 2-mercaptoethanol for Reaction (1), substituting 50 mM triethanolamine-Cl, pH 8.9, for the phosphate buffer, 50 mM (NH4)2SO4 for glutamine, 2 mM thioglycerol for 2-mercaptoethanol, and increasing the starting chorismate level to 0.3 mM for Reaction (2). Incubation was at 23 °. Fluorometer settings were optimized for the anthranilate standard (excitation about 325 nm and emission about 400 nm) and a unit of activity was the appearance of 1 /xmol of anthranilate per minute. Protein was assayed conventionally. 8 Purification Procedure, Method 16 Cells were broken by sonic oscillation after suspension at 1:1.5 (w/v) in 20 mM Tfis-C1, pH 7.8, 13 mM 2-mercaptoethanol, and 10 mM MgCl2 (Buffer A). Centrifugation at 40,000 g for 30 rain gave the crude extract, which was stable when frozen at - 1 5 °. Eight hundred milliliters of crude extract was treated twice with 8 mg DNase I and 760 mg MgCI2 for 20 min at room temperature, then heated to 55 ° for 8 min, chilled, and centrifuged at 25,000 g for 20 rain. Solid (NH,)2SO4 was added to 30% saturation, the resulting precipitate was removed, and the supernatant adjusted to 50% saturation with (NH4)2SO4. Precipitated protein was centrifuged and dissolved in 40 ml of 20 mM Tris-Cl, pH 7.8, 13 mM 2-mercaptoethanol, and 0.1 mM EDTA (Buffer B). This preliminary 2.6-fold purification of the aft complex was followed directly by gel filtration to separate the subunits. The 30-50% fraction was passed through a 3 liter (80 × 7 cm) column of Sephadex G-100 equilibrated with Buffer B at a flow rate of 1 ml/min. The a subunit, eluting between 1400 and 1850 ml, was concentrated by ultrafiltration (Aminco UM-10 diaflow membrane). The/3 subunit, cluting between 2400 and 3900 ml, was concentrated similarly. Both pools were stabilized by the addition of MgCl2 to a final concentration of 10 raM. The fraction containing a subunit was placed on a 50 × 4 cm column of DEAE-cellulose cquilibratcd with Buffer A. After washing with 1 liter amounts of Buffer A containing 70 and 140 mM KC1, a linear gradient of 140-300 mM KC1 in Buffer A was begun. Free a subunit clutcd near the end of the 140 mM KC1 wash and the small amount of a/3 complex followed it at about 220 mM KC1. The fractions containing free a subunit were pooled, dialyzed against Buffer A, and concentrated to 5 ml by 8 0 . H. Lowry, N. J. Roscbrough, A. L. Farr, and R. J. Randall J. Biol. Chem. 152, 293 (1951); or this series, Vol. 3 [73].
[38]
ANTHRANILATE SYNTHASE FROM PSEUDOMONADS
303
ultrafiltration. The final purification of a subunit was accomplished by preparative polyacrylamide electrophoresis of 1.2-ml aliquots in a Canalco apparatus. The sample containing 20% sucrose and 0.1% ravin mononucleotide added as a marker was added to the 5-cm-diameter column equilibrated in 35 mM Tris-10 mM glycine buffer at pH 7.8 containing 15 mM 2-mercaptoethanol. Electrophoresis was accomplished at 50 mA for 8 hr. Fractions of 4.8 ml were collected at the anode. Those with a specific activity greater than 4.5 were pooled and concentrated to 5 ml as described above. The Sephadex G-100 fractions containing/3 subunit were further purified by chromatography on a 50 x 4 cm DEAE-cellulose column equilibrated with Buffer A. Upon elution with a linear gradient of 0-140 mM KCI, two protein peaks were observed, the second, smaller one containing the/3 subunit activity. The most active fractions were pooled, dialyzed against Buffer A, and concentrated to 5 ml by ultrafiltration. Final purification of the/3 subunit was accomplished by chromatography on a column of calcium phosphate gel prepared according to Seligman e t al. 9 Just prior to addition of the sample, the column was washed with 10 volumes of cold distilled water. Elution was with washes of 1 and 3 mM potassium phosphate, both at pH 7.5. Two major protein peaks emerged, with the/3 subunit activity confined to the first. Active fractions were combined and dialyzed against Buffer A. The course of purification is shown in Table I. The final fractions showed a single protein band in polyacrylamide gel electrophoresis tubes loaded with 50-100/zg of protein and stained with amido schwartz. When unstained gels were sliced and eluted, only those slices corresponding to the stained bands contained a or/3 subunit activity. Molecular weight estimates by ultracentrifugation were 63,400 -+ 2600 for the a subunit and 18,000 -+ 1100 for the/3 subunit. Purification Procedure, Method II 7 All steps were carried out at 2-5 °. Cells were suspended at 1:1.5 (w/v) in 40 mM sodium borate, pH 7.8, 20 mM 2-mercaptoethanol, 10 mM MgCI2, 5 mM L-glutamine, 20 mM EDTA, and 70/zg DNase I/ml and disrupted in a French press at 12,000 psi. The broken cell suspension was diluted with an equal volume of buffer solution and centrifuged at 100,000 g for 60 min. Solid (NH4)2SO4 (285 g/liter) was added to the supernatant, maintaining the pH at 7.8 by the addition of 8% NH4OH. After standing 45 min the suspension was centrifuged at 9000 g for 45 rain. The precipitate was suspended in Buffer A (10 mM sodium borate, pH 7.8, 20 mM 29 H. W. Seligman, G. A. Wieczorek, and B. C. Turner, Anal. Biochem. 13, 402 (1965).
304
B I O S Y N T H E S OF I S THE AROMATIC AMINO ACIDS
[38]
TABLE I PURIFICATION OF P. putida ANTHRANILATE SYNTHASE BY METHOD 16 Anthranilate synthase ~
Fraction Crude extract 55° Supernatant 30-50% (NH4)2SO4 ~t Subunit Sephadex G-100 DEAE-cellulose Electrophoresis /3 Subunit Sephadex G-100 DEAE-Sephadex Calcium phosphate
Volume (ml)
Protein (g)
a Subunit b
/3 Subunit c
Specific Activity Yield activity (units) (%) (units/mg)
Specific Activity Yield activity (units) (%) (units/mg)
820 820 70
31 19 12
1720 1700 1900
100 99 110
0.06 0.09 0.16
1760 1700 1950
100 96 111
0.06 0.09 0.16
450 5 5
2.6 0.45 0.10
1290 1120 470
75 65 25
0.50 2.5 4.7
150 0 0
8 0 0
0.06 0 0
1500 120 I l0
1.4 0.154 0.042
0 0 0
0 0 0
1420 1000 720
80 57 41
1.01 6.5 17.0
0 0 0
a Determined as described in the text. One unit of activity is the conversion of 1/zmol of chorismate and glutamine to anthranilate per minute at 37°. b Determined with the addition of a 5-fold excess of the purified/3 subunit. c Determined with the addition of a 5-fold excess of the purified a subunit.
mercaptoethanol, 10 mM MgC12, and 1 mM EDTA), dialyzed thoroughly against 25 volumes of this buffer, and centrifuged at 27,000 g for 45 min to remove insoluble material. The supernatant from the previous step was diluted 1:3 with Buffer B (Buffer A + 5 mM L-glutamine) and applied to a 40 × 2.5 cm column of DEAE-cellulose equilibrated with Buffer B. The column was washed with 400 ml of Buffer B, then with 800 ml of Buffer B containing 30 mM MgCl2. Elution was with a linear 1.2 liter gradient of 30-150 mM MgCI2 in Buffer B. The fractions containing the a/3 complex were precipitated with 430 g/ liter of ( N H 4 ) 2 S O 4 , centrifuged, suspended in 45 ml of 20 mM Tris-C1, pH 7.8, 20 mM 2-mercaptoethanol, and 5 mM EDTA, and dialyzed against the same buffer containing 1 mM L-tryptophan and 3 M KCI (Buffer C). Enzyme in Buffer C exhibits reduced activity; it can be reactivated by dialysis against Buffer A.) The dialyzed enzyme was applied to the first of a tandem pair of Sephadex G-100 columns (100 × 5 cm each) equilibrated with Buffer C and eluted using upward flow. Those fractions containing the a subunit were pooled and dialyzed against 20 mM Tris-C1, pH 7.8, 10 mM 2-
[38]
305
ANTHRANILATE SYNTHASE FROM PSEUDOMONADS T A B L E II PURIFICATION OF P. putida ANTHRANILATE SYNTHASE BY METHOD 117 Anthranilate s y n t h a s e Reaction (1)
Fraction Crude extract (NH4hSO4 Ppt. DEAE-cellulose /3 Subunit S e p h a d e x G-100 DEAE-cellulose
Reaction (2)
Volume (ml)
Protein (g)
Activity (units)
Specific activity (units/mg)
Activity (units)
Specific activity (units/rag)
2280 550 210
25.4 36.9 14.8
2100 2030 1840
0.037 0.10 0.47
1670 1760 1610
0.029 0.086 0.41
---
---
415 16
0.43 0.18
1010b 670 b
5.7 23.0
a A s s a y e d as described in the text without the addition of purified a or/3 subunit. One unit o f activity is the conversion o f l /~mol of chorismate to anthranilate per minute at 23 ° . b Determined with the addition o f e x c e s s c~ subunit. T h e s e fractions have no activity in its absence.
mercaptoethanol, 10 mM MgC12, and 1 mM EDTA. The fractions containing the fl subunit were pooled separately and extensively dialyzed against l0 liters of Buffer D (10 mM sodium borate, pH 7.8, 20 mM 2-mercaptoethanol, 2.5 mM EDTA), changing the dialysis buffer 5 times. The ct subunit could be purified further by chromatography on hydroxylapatite. This removed the small amount of residual activity in reaction 1 and in the experiment shown in Table II resulted in a preparation containing 660 units of Reaction (2) activity with a specific activity of 2.21 units/ mg, estimated to be 60% pure. 7 Stored in 20 mM Tris-C1, pH 7.8, 10 mM 2-mercaptoethanol, 10 mM MgCI2, 1 mM EDTA, and 30% glycerol it retained full activity for 3 months at 4°, after which time activity gradually declined. The dialyzed fractions containing the/3 subunit were applied to a 30 x 1.5 cm column of DEAE-cellulose equilibrated with Buffer D. After washing with 700 ml of the same buffer the enzyme was eluted with a 500 ml linear gradient of 100-200 mM KCI in Buffer D. Fractions of constant specific activity were pooled and concentrated using a small (6 x 1 cm) DEAE-cellulose column. The/3 subunit was stored as eluted at 2-5 °. Its activity declined upon storage, but was nearly fully restored by dialysis against 50 mM potassium phosphate, pH 7.8, 20 mM 2-mercaptoethanol, l0 mM EDTA for 5 hr followed by dialysis against 50 mM potassium
306
BIOSYNTHESIS
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.
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
[38]
The trp genes of other tryptophan-synthesizing eukaryotes have not yet been thoroughly analyzed. The tendency for gene fusion exhibited by the fungi appears to have been carried further in Euglena gracilis, where all the enzymes of the pathway except AS are incorporated into a single, large polypeptide chain over 200,000 in molecular weight. 3j It should not be assumed that this situation holds for all photosynthetic eukaryotes, however, for both the green algae and plants seem to have all the activities of the pathway on separate molecules, and their TS has a and /3 subunits resembling the bacterial o n e s ) 2 Gene structure has not been thoroughly analyzed in these cases, however. 3~ j. C. L a r a and S. E. Mills, J. Bacteriol. 110, 1100 (1972). 32 j. C h e n and W. G. Boll, Can. J. Bot. 50, 587 (1972); R. T. N a g a n o and T. C. Moore, Arch. Biochem. Biophys. 149, 402 (1972); C. N. Hankins, M. T. Largen, and S. E. Mills, Plant Physiol. 57, 101 (1976).
[38] A n t h r a n i l a t e S y n t h a s e f r o m F l u o r e s c e n t P s e u d o m o n a d s
By IRVING P. CRAWFORD Anthranilate synthase, the first enzyme specific to tryptophan synthesis, has been intensively studied in a number of organisms, l The enzyme normally catalyzes Reaction (1). C h o r i s m a t e + L-glutamine ~ anthranilate + pyruvate + L-glutamate
(1)
In Pseudomonas putida and Pseudomonas aeruginosa the enzyme has an 0//3 subunit structure. The larger a subunit (component I in the nomenclature of Ito and Yanofsky2), can catalyze Reaction (2) independently. C h o r i s m a t e + NH3 ~ anthranilate + pyruvate •
(2)
The smaller/3 subunit (component II) contributes a glutamine binding site to the complex; it belongs to a family of glutamine amidotransferase molecules associated with the anthranilate and p-aminobenzoate synthases from a variety of bacteria and fungi) Because the gene for this small subunit is not fused to other pathway components and the enzyme does not self-aggregate to the 0/2/32 form, anthranilate synthase from fluorescent 1 H. Zaikin, Adv. Enzymol. 38, I (1973); in "Multifunctional Proteins" (H. Bisswanger and E. Schmincke-Ott, eds.), p. 123. Wiley, N e w York, 1980. 2 j. Ito and C. Y a n o f s k y , J. Biol. Chem. 241, 4112 (1966). 3 j. B. Kaplan, W. K. Merkel, a n d B. P. Nichols, J. Mol. Biol. 183, 327 (1985).
METHODS IN ENZYMOLOGY, VOL. 142
Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.
[38]
ANTHRANILATE SYNTHASE FROM PSEUDOMONADS
301
pseudomonads is the smallest and simplest form of the enzyme that has been studied intensively. ~Despite its structural simplicity and small size, however, the enzyme shows typical feedback inhibition by L-tryptophan. As in other, better studied instances 4 the a subunit has been shown to contain the tryptophan binding site) Two procedures have been devised for the purification of P. p u t i d a anthranilate synthase. Both rely on controlling the reversible dissociation of the subunits and employ fractionations first for the a/3 complex, then for the individual subunits. Although Queener et al. 6 achieved purity of both components, reporting a yield of 25% for the a and 41% for the/3 subunit, Goto et al. 7 had to revise this procedure considerably to obtain pure/3 subunit. Their procedure gave pure/3 subunit in 32% yield, with a subunit being only 60% pure but essentially free of/3 subunit. Both procedures will be described. Growth of Cells The a and/3 subunits of anthranilate synthase are derepressed coordinately when an auxotroph blocked later in the pathway is starved for tryptophan. The auxotroph used by Queener et al., 6 PpG281 trpF21, was a slightly leaky mutant blocked in the third step of the pathway, while that used by Goto et al. 7 was trpC621, blocked in the fourth step. As both mutants had the same P. p u t i d a parent, it seems unlikely that strain differences affected the purification of the enzyme. Because of variation in the assay conditions used by the two laboratories it is not certain that equivalent levels of derepression were obtained in the two studies, however. Assay Procedures Both methods employed the continuous fluorometric assay for anthranilate formation that has been described in earlier contributions in this series (Vol. XVIIA [46, 47, 48, 48a]; Vol. 113 [37]). Activity levels were high enough so that even crude extracts could be assayed directly in the fluorometer cuvette. Queener et al. used 25 mM Tris-C1, pH 7.8, 5 mM MgCI2, 5 mM L-glutamine, 0.25 mM chorismate, and 13 mM 2-mercaptoethanol for Reaction (1), substituting 0.2 M glycine, pH 8.8, for the Tris 4 H. Nagano and H. Zalkin, J. Biol. Chem. 245, 3097 (1970). 5 S. F. Queener and I. C. Gunsalus, Proc. Natl. Acad. Sci. U.S.A. 67, 1225 (1970). 6 S. F. Queener, S. F. Queener, J. R. Meeks, and 1. C. Gunsalus, J. Biol. Chem. 2,48, 151 (1973). 7 y . Goto, H. Zalkin, P. S. Keim, and R. L. Heinrikson, J. Biol. Chem. 25L 941 (1976).
302
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
[38]
buffer and 0.3 M (NH4)2 SO4 for glutamine to measure Reaction (2). Their reaction mixture was maintained at 37°. Goto et al. used 50 mM potassium phosphate (pH 7.4), 20 mM L-glutamine, l0 mM MgC12, 0.1 mM chorismate, and 25 mM 2-mercaptoethanol for Reaction (1), substituting 50 mM triethanolamine-Cl, pH 8.9, for the phosphate buffer, 50 mM (NH4)2SO4 for glutamine, 2 mM thioglycerol for 2-mercaptoethanol, and increasing the starting chorismate level to 0.3 mM for Reaction (2). Incubation was at 23 °. Fluorometer settings were optimized for the anthranilate standard (excitation about 325 nm and emission about 400 nm) and a unit of activity was the appearance of 1 /xmol of anthranilate per minute. Protein was assayed conventionally. 8 Purification Procedure, Method 16 Cells were broken by sonic oscillation after suspension at 1:1.5 (w/v) in 20 mM Tfis-C1, pH 7.8, 13 mM 2-mercaptoethanol, and 10 mM MgCl2 (Buffer A). Centrifugation at 40,000 g for 30 rain gave the crude extract, which was stable when frozen at - 1 5 °. Eight hundred milliliters of crude extract was treated twice with 8 mg DNase I and 760 mg MgCI2 for 20 min at room temperature, then heated to 55 ° for 8 min, chilled, and centrifuged at 25,000 g for 20 rain. Solid (NH,)2SO4 was added to 30% saturation, the resulting precipitate was removed, and the supernatant adjusted to 50% saturation with (NH4)2SO4. Precipitated protein was centrifuged and dissolved in 40 ml of 20 mM Tris-Cl, pH 7.8, 13 mM 2-mercaptoethanol, and 0.1 mM EDTA (Buffer B). This preliminary 2.6-fold purification of the aft complex was followed directly by gel filtration to separate the subunits. The 30-50% fraction was passed through a 3 liter (80 × 7 cm) column of Sephadex G-100 equilibrated with Buffer B at a flow rate of 1 ml/min. The a subunit, eluting between 1400 and 1850 ml, was concentrated by ultrafiltration (Aminco UM-10 diaflow membrane). The/3 subunit, cluting between 2400 and 3900 ml, was concentrated similarly. Both pools were stabilized by the addition of MgCl2 to a final concentration of 10 raM. The fraction containing a subunit was placed on a 50 × 4 cm column of DEAE-cellulose cquilibratcd with Buffer A. After washing with 1 liter amounts of Buffer A containing 70 and 140 mM KC1, a linear gradient of 140-300 mM KC1 in Buffer A was begun. Free a subunit clutcd near the end of the 140 mM KC1 wash and the small amount of a/3 complex followed it at about 220 mM KC1. The fractions containing free a subunit were pooled, dialyzed against Buffer A, and concentrated to 5 ml by 8 0 . H. Lowry, N. J. Roscbrough, A. L. Farr, and R. J. Randall J. Biol. Chem. 152, 293 (1951); or this series, Vol. 3 [73].
[38]
ANTHRANILATE SYNTHASE FROM PSEUDOMONADS
303
ultrafiltration. The final purification of a subunit was accomplished by preparative polyacrylamide electrophoresis of 1.2-ml aliquots in a Canalco apparatus. The sample containing 20% sucrose and 0.1% ravin mononucleotide added as a marker was added to the 5-cm-diameter column equilibrated in 35 mM Tris-10 mM glycine buffer at pH 7.8 containing 15 mM 2-mercaptoethanol. Electrophoresis was accomplished at 50 mA for 8 hr. Fractions of 4.8 ml were collected at the anode. Those with a specific activity greater than 4.5 were pooled and concentrated to 5 ml as described above. The Sephadex G-100 fractions containing/3 subunit were further purified by chromatography on a 50 x 4 cm DEAE-cellulose column equilibrated with Buffer A. Upon elution with a linear gradient of 0-140 mM KCI, two protein peaks were observed, the second, smaller one containing the/3 subunit activity. The most active fractions were pooled, dialyzed against Buffer A, and concentrated to 5 ml by ultrafiltration. Final purification of the/3 subunit was accomplished by chromatography on a column of calcium phosphate gel prepared according to Seligman e t al. 9 Just prior to addition of the sample, the column was washed with 10 volumes of cold distilled water. Elution was with washes of 1 and 3 mM potassium phosphate, both at pH 7.5. Two major protein peaks emerged, with the/3 subunit activity confined to the first. Active fractions were combined and dialyzed against Buffer A. The course of purification is shown in Table I. The final fractions showed a single protein band in polyacrylamide gel electrophoresis tubes loaded with 50-100/zg of protein and stained with amido schwartz. When unstained gels were sliced and eluted, only those slices corresponding to the stained bands contained a or/3 subunit activity. Molecular weight estimates by ultracentrifugation were 63,400 -+ 2600 for the a subunit and 18,000 -+ 1100 for the/3 subunit. Purification Procedure, Method II 7 All steps were carried out at 2-5 °. Cells were suspended at 1:1.5 (w/v) in 40 mM sodium borate, pH 7.8, 20 mM 2-mercaptoethanol, 10 mM MgCI2, 5 mM L-glutamine, 20 mM EDTA, and 70/zg DNase I/ml and disrupted in a French press at 12,000 psi. The broken cell suspension was diluted with an equal volume of buffer solution and centrifuged at 100,000 g for 60 min. Solid (NH4)2SO4 (285 g/liter) was added to the supernatant, maintaining the pH at 7.8 by the addition of 8% NH4OH. After standing 45 min the suspension was centrifuged at 9000 g for 45 rain. The precipitate was suspended in Buffer A (10 mM sodium borate, pH 7.8, 20 mM 29 H. W. Seligman, G. A. Wieczorek, and B. C. Turner, Anal. Biochem. 13, 402 (1965).
304
B I O S Y N T H E S OF I S THE AROMATIC AMINO ACIDS
[38]
TABLE I PURIFICATION OF P. putida ANTHRANILATE SYNTHASE BY METHOD 16 Anthranilate synthase ~
Fraction Crude extract 55° Supernatant 30-50% (NH4)2SO4 ~t Subunit Sephadex G-100 DEAE-cellulose Electrophoresis /3 Subunit Sephadex G-100 DEAE-Sephadex Calcium phosphate
Volume (ml)
Protein (g)
a Subunit b
/3 Subunit c
Specific Activity Yield activity (units) (%) (units/mg)
Specific Activity Yield activity (units) (%) (units/mg)
820 820 70
31 19 12
1720 1700 1900
100 99 110
0.06 0.09 0.16
1760 1700 1950
100 96 111
0.06 0.09 0.16
450 5 5
2.6 0.45 0.10
1290 1120 470
75 65 25
0.50 2.5 4.7
150 0 0
8 0 0
0.06 0 0
1500 120 I l0
1.4 0.154 0.042
0 0 0
0 0 0
1420 1000 720
80 57 41
1.01 6.5 17.0
0 0 0
a Determined as described in the text. One unit of activity is the conversion of 1/zmol of chorismate and glutamine to anthranilate per minute at 37°. b Determined with the addition of a 5-fold excess of the purified/3 subunit. c Determined with the addition of a 5-fold excess of the purified a subunit.
mercaptoethanol, 10 mM MgC12, and 1 mM EDTA), dialyzed thoroughly against 25 volumes of this buffer, and centrifuged at 27,000 g for 45 min to remove insoluble material. The supernatant from the previous step was diluted 1:3 with Buffer B (Buffer A + 5 mM L-glutamine) and applied to a 40 × 2.5 cm column of DEAE-cellulose equilibrated with Buffer B. The column was washed with 400 ml of Buffer B, then with 800 ml of Buffer B containing 30 mM MgCl2. Elution was with a linear 1.2 liter gradient of 30-150 mM MgCI2 in Buffer B. The fractions containing the a/3 complex were precipitated with 430 g/ liter of ( N H 4 ) 2 S O 4 , centrifuged, suspended in 45 ml of 20 mM Tris-C1, pH 7.8, 20 mM 2-mercaptoethanol, and 5 mM EDTA, and dialyzed against the same buffer containing 1 mM L-tryptophan and 3 M KCI (Buffer C). Enzyme in Buffer C exhibits reduced activity; it can be reactivated by dialysis against Buffer A.) The dialyzed enzyme was applied to the first of a tandem pair of Sephadex G-100 columns (100 × 5 cm each) equilibrated with Buffer C and eluted using upward flow. Those fractions containing the a subunit were pooled and dialyzed against 20 mM Tris-C1, pH 7.8, 10 mM 2-
[38]
305
ANTHRANILATE SYNTHASE FROM PSEUDOMONADS T A B L E II PURIFICATION OF P. putida ANTHRANILATE SYNTHASE BY METHOD 117 Anthranilate s y n t h a s e Reaction (1)
Fraction Crude extract (NH4hSO4 Ppt. DEAE-cellulose /3 Subunit S e p h a d e x G-100 DEAE-cellulose
Reaction (2)
Volume (ml)
Protein (g)
Activity (units)
Specific activity (units/mg)
Activity (units)
Specific activity (units/rag)
2280 550 210
25.4 36.9 14.8
2100 2030 1840
0.037 0.10 0.47
1670 1760 1610
0.029 0.086 0.41
---
---
415 16
0.43 0.18
1010b 670 b
5.7 23.0
a A s s a y e d as described in the text without the addition of purified a or/3 subunit. One unit o f activity is the conversion o f l /~mol of chorismate to anthranilate per minute at 23 ° . b Determined with the addition o f e x c e s s c~ subunit. T h e s e fractions have no activity in its absence.
mercaptoethanol, 10 mM MgC12, and 1 mM EDTA. The fractions containing the fl subunit were pooled separately and extensively dialyzed against l0 liters of Buffer D (10 mM sodium borate, pH 7.8, 20 mM 2-mercaptoethanol, 2.5 mM EDTA), changing the dialysis buffer 5 times. The ct subunit could be purified further by chromatography on hydroxylapatite. This removed the small amount of residual activity in reaction 1 and in the experiment shown in Table II resulted in a preparation containing 660 units of Reaction (2) activity with a specific activity of 2.21 units/ mg, estimated to be 60% pure. 7 Stored in 20 mM Tris-C1, pH 7.8, 10 mM 2-mercaptoethanol, 10 mM MgCI2, 1 mM EDTA, and 30% glycerol it retained full activity for 3 months at 4°, after which time activity gradually declined. The dialyzed fractions containing the/3 subunit were applied to a 30 x 1.5 cm column of DEAE-cellulose equilibrated with Buffer D. After washing with 700 ml of the same buffer the enzyme was eluted with a 500 ml linear gradient of 100-200 mM KCI in Buffer D. Fractions of constant specific activity were pooled and concentrated using a small (6 x 1 cm) DEAE-cellulose column. The/3 subunit was stored as eluted at 2-5 °. Its activity declined upon storage, but was nearly fully restored by dialysis against 50 mM potassium phosphate, pH 7.8, 20 mM 2-mercaptoethanol, l0 mM EDTA for 5 hr followed by dialysis against 50 mM potassium
306
BIOSYNTHESIS
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.