The first committed step of aromatic amino acidbiosynthesis in Salmonella ... aroF, the structural gene for the tyrosine-inhibitable isoenzyme, ..... Glyoxylic acid.
JOURNAL OF BACTERIOLOGY, Dec. 1975, P. 1312-1320 Copyright 0 1975 American Society for Microbiology
Vol. 124, No. 3 Printed in U.S.A.
3-Deoxy-D-Arabino-Heptulosonic Acid 7-Phosphate Synthase Mutants of Salmonella typhimurium ALBERT B. DELEO' AND D. B. SPRINSON* Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, New York 10032 Received for publication 19 September 1975
The first committed step of aromatic amino acid biosynthesis in Salmonella typhimurium was shown to be catalyzed by three isoenzymes of 3-deoxy-Darabino-heptulosonic acid 7-phosphate (DAHP) synthase. Mutations in each of the genes specifying the isoenzymes were isolated and mapped. aroG, the structural gene for the phenylalanine-inhibitable isoenzyme, was linked to gal, and aroH, the structural gene for the tryptophan-inhibitable isoenzyme, was linked to aroE. aroF, the structural gene for the tyrosine-inhibitable isoenzyme, was linked to pheA and tyrA, which specify the phenylalanine- and tyrosinespecific branch-point enzymes, respectively. The phenylalanine-inhibitable isoenzyme was the predominant DAHP synthase in wild-type cells, and only the tyrosine-inhibitable isoenzyme was completely repressed, as well as inhibited, by low levels of its allosteric effector. The DAHP synthase isoenzymes were separated by chromatography on diethylaminoethyl-cellulose with a phosphate gradient which contained enolpyruvate phosphate to protect the otherwise unstable phenylalanine-inhibitable isoenzyme. No cross-inhibition of either the tyrosine- or phenylalanine-inhibitable isoenzyme was observed at inhibitor concentrations up to 1 mM. The tryptophan-inhibitable isoenzyme was partially purified from extracts of a strain lacking the other two isoenzymes and shown to be inhibited about 30% by 1 mM tryptophan. A preliminary study of interference by tryptophan in the periodate-thiobarbiturate assay for DAHP suggested a combined effect of tryptophan and erythrose 4-phosphate, or an aldehydic compound resulting from degradation of erythrose 4-phosphate by periodate.
Regulation of aromatic amino acid biosynthesis occurs primarily at the first reaction and at the branch-point enzymes after chorismic acid (Fig. 1; 4, 14, 17, 26). In many bacterial species and in Neurospora, the first committed step of the pathway, formation of 3-deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP), is catalyzed by three isoenzymes under the control of tyrosine, phenylalanine, and tryptophan. In the present report we describe isolation and mapping of three types of mutants, each lacking one of the three isoenzymes. A mutant strain containing only the tryptophan-sensitive isoenzymes was also isolated. Repression and feedback inhibition of DAHP synthase (EC 188.8.131.52) activities were studied in these strains, and optimum conditions were found for separation of the isoenzymes by chromatography on diethylaminoethyl (DEAE)-cellulose. (This report is in partial fulfillment for the
degree of Doctor of Philosophy, submitted by A.B.D., February 1970.)
MATERIALS AND METHODS Organisms. The bacterial strains employed were derivatives of Salmonella typhimurium LT 2 and are listed in Table 1. Strain PM316 (gal-205ara-9) was obtained from P. Margolin, and all other strains were from the stock collection of the late M. Demerec. Phage P22 was obtained from R. Rudner. Culture media. Minimal medium contained the salts solution of Vogel and Bonner (28) supplemented with 0.4% glucose (sterilized separately as a 40% solution). Enriched minimal medium, used in isolation of mutants, contained the 17 non-aromatic amino acids (8 x 10-I M each), adenine and uracil (20 Ag per ml), and thiamine, riboflavin, nicotinic acid, pyridoxine, pantothenic acid, and biotin (2 gg per ml). Minimal medium M-9 (1) contained either 1% galactose and 0.04% eosine plus 0.0065% methylene blue as indicators to distinguish between gal+ and gal- organisms, or 0.2% galactose without indicators. Culture 'Present address: Memorial Sloan-Kettering Cancer Cen- plates were prepared by adding 1.5% agar (Difco) to ter, New York, N.Y. 10021. the liquid media. Media supplemented with aromatic 1312
VOL. 124, 1975 C02H it
F, CH2 GH CHO
three aromatic amino acids (0.2 mM each) and with
'< oroC_ OA-J\OH HO"OH
FIG. 1. Biosyvnthesis of chorismic acid and its conversion to arormnatic end products. DHQa 3-Dehydroquinate; Dh IS, 3-dehydroshikimate,S-3-P, shikimate 3-phosphc ate; PABA, 4-aminobenzoate; PHBAi 4-hydroxybenzo bate. The enzymes specified by the indicated genes iare: aroF, tyrosine-inhibited DAHP synthase; aroG, phenylalanine-inhibited DAHP synthase; aroH, tr -yptophan-inhibited DAHP synthase; aroB, 3-dehydr( yquinate synthase; aroE, dehydroqui nase; aroC, de qhydroshikimate reductase, aroA 5enolpyruvl-shik hmate-3-phosphate synthase; aroD, chorismate synt thase. ae
1. Bacterial strains
levant Rel gen otype
Lacking the tyrosineinhibitable DAHP synthase
Derivative of SD101 Lacking the phenylalanine-inhibitable DAHP synthase Lacking the tryptophaninhibitable DAHP synthase
gal-*205ara-9 tyrA13 pheA3 arolE138
Lacking the phenylalanine- and tyrosineinhibitable DAHP synthase isoenzymes
Tyrosine auxotroph; lacks prephenate dehydrogenase (7) Phenylalanine auxotroph; lacks prephenate dehydratase (7) Lacks dehydroquinase (17)
0.1 mM of each, unless otherwise specified. Isolation of mutants. Mutagenesis by N-methylN'-nitro-N-nitrcDsoguanidine was carried out essentially as describ ed by Adelberg et al. (2). Phenotypic expression was allowed to proceed by incubating the washed mutage] nized cells for 4 h at 37 C with shaking in enriched min Limal medium supplemented with the amino acids conitained
4-hydroxybenzoate and 4-aminobenzoate (0.01 mM
each). The cells were removed by centrifugation, washed with minimal medium, and resuspended at a dilution of approximately 107 cells per ml in enriched minimal medium containing pairs of the three aromatic amino acids (0.2 mM). Penicillin (300 USP units per ml) was added, and the culture was allowed to incubate for 4 h without shaking at 37 C. The cells were
DAHP SYNTHASE MUTANTS OF SALMONELLA
by centrifugation, resuspended
mal medium, and plated on minimal agar. After 24 h of incubation at 37 C, the plates were replicated on minimal medium plates supplemented with pairs of aromatic
after 24 h at 37 C.
Transduction. Lysates were prepared according to Adams (1) by infecting donor cells grown to log phase
in nutrient broth with P22 phage
units and 2 x 108 cells per ml) and by incubating for at least 8 h at 37 C with vigorous shaking. Recipient bacteria,
were infected with phage (1010 phage and 2
min at 37 C without shaking, and plated on the medium indicated.
per ml), incubated for 6
Preparation of cell extracts. Cells were grown
with vigorous shaking at 37 C to late log phase, harvested by centrifugation in the cold, and washed once
with cold 0.05 M potassium phosphate buffer,
pH 7.0. (Cell extracts were prepared or the cells were frozen at -15 C). Cells were resuspended in the same buffer (1 g of packed wet cells per 4 ml), chilled in ice, and disrupted in a M.S.E. 60-W ultrasonic disintegrator for 12 periods of 15 s each alternating with 15-s rest periods. The suspension was centrifuged at 43,000 x g for 1 h at 4 C, and clear supernatant solutions were analyzed the same day. DEAE-cellulose chromatography. DEAE-cellulose was washed several times with 0.01 M potassium phosphate buffer, pH 6.8, kept overnight in the same buffer containing 2% sodium chloride and washed several times with buffer to remove salt. Jacketed columns (1.6 by 25 cm, at 4 C) of DEAE-cellulose (carefully treated to remove fine particles) were washed with 1 liter of buffer and loaded with cell extract (1 g of protein per 150 cm' of adsorbent). Elution was carried out at approximately 30 ml per h with a linear gradient between 500 ml of 0.01 M potassium phosphate buffer, pH 6.8, and 500 ml of 0.5 M potassium phosphate, pH 6.8. Fractions of 5.0 ml were collected. Certain extracts were chromato-
graphed with enolpyruvate-P either at a constant concentration of 1 mM or in a linear gradient from 0.0 to 1.0 mM (see below). Materials. Monocyclohexylammonium enolpyruvate-P was prepared according to Clark and Kirby (6) and converted to the potassium salt. Erythrose-4-P was prepared according to Ballou and MacDonald (3). DEAE-cellulose (Selectacel type 20) was obtained from Schleicher & Schuell Co. The following compounds were gifts: a-methyl-DL-tryptophan from P. Feigelson, 2-methyl-DL-tryptophan from H. N. Rydon, 1-methyl-DL-tryptophan from E. Leete, 2,3dihydro-L-tryptophan from B. Witkop, and glycolaldehyde-2-P from C. E. Ballou. All other chemicals
DELEO AND SPRINSON
were obtained commercially and used without purification. Analytical procedures. Enolpyruvate-P was assayed with pyruvate kinase and lactate dehydrogenase as described by Kornberg and Pricer (19). The concentration of enolpyruvate-P in column eluants was determined by hydrolysis in 0.3 N HCl at 100 C for 10 min; protein was removed by centrifugation, and the supernatant solutions were treated with 2,4-dinitrophenylhydrazine (13). DAHP synthase activity in cell extracts was determined at 37 C as previously described (17) except that 60 mM tris(hydroxymethyl)aminomethane maleate buffer, pH 6.8, was used. Erythrose-4-P was determined with DAHP synthase in the presence of an excess of enolpyruvate-P. Protein was determined by the method of Lowry et al. (20).
0.2% galactose, and a gal- mutant was isolated. The resulting strain, SD103, was transduced to Gal+ with phage grown on strain SD201 (aroG9gal+), and transductants were scored for inability to grow on minimal medium M-9 containing 0.2% galactose and 0.1 mM tryptophan. A transductant strain SD401 (aroF3aroG9) had only the tryptophan-inhibitable DAHP synthase, as shown below. Regulation of DAHP synthase isoenzymes. Strains with two of the three DAHP synthase isoenzymes exhibited normal growth behavior TABLE 2. Linkage of DAHP synthase genes No. Recipient Donor Selected transDonor genotype genotype phenotype ductants characterma ined
RESULTS Isolation and genetic analysis of DAHP synthase mutants. Isolation of mutants lacking tyrA3 aroF3 180 AroF-c (70) Tyr+b one of the three DAHP synthase isoenzymes was pheA3 aroF3 Phe+b 342 AroF- (58) aroHi Aro+5 174 based on the premise that they exhibited their aroE138 AroH-d (38) AroG+e gal-205 117 Gal - (45) phenotype only in the presence of a pair of aroG9 amino acids which repressed and inhibited the a All colonies were scored after incubation for 24 h at 37 C. unaltered two isoenzymes. This experimental bTransductants were plated on minimal medium and design is analogous to that used by Patte and incubated 48 h at 37 C. c Transductants were plated on minimal medium and Cohen (22) to isolate strains of Escherichia coli minimal medium supplemented with phenylalanine and lacking one of the aspartokinase isoenzymes. tryptophan. Thus, mutants with lesions in aroF, the strucd Transductants were plated on minimal medium and tural gene for the tyrosine-inhibitable isoen- minimal medium supplemented with tyrosine and phenylalazyme, were selected by scoring for inability to nine. eTransductants were plated on minimal medium supplegrow on minimal medium supplemented with mented with tyrosine and tryptophan and incubated for 48 h phenylalanine and tryptophan. Similarly, mu- at 37 C. tants with lesions in aroG, the structural gene ' Transductants were plated on eosin methylene blue for the phenylalanine-inhibitable isoenzyme, medium supplemented with tyrosine and tryptophan. were unable to grow in the presence of tyrosine 0 and tryptophan, and mutants with lesions in o cr aroH were unable to grow in the presence of Q 0 phenylalanine and tyrosine. While this work was in progress, aroF in E. coli K-12 was found to be linked to pheA and tyrA, the structural genes for the phenylalanine and tyrosine branch-point enzymes, respectively, whereas aroG was found to be linked with gal (4, 29). Furthermore, aroH was found to be linked with aroB, which in E. coli specifies dehydroquinase (29). Linkage of DAHP synthase genes in Salmonella, therefore, was studied on the basis of homology with respective E. coli genes. aroF was found to be 70% co-transducible with tyrA and 58% co-transducible with pheA, indicating close linkage of aroF with these two aromatic loci (Table 0 2). aroG was found to be 45% co-transducible CD with gal, and aroH 38% co-transducible with 0 aroE (Fig. 2). Strain SD101 (aroF3) was mutagenized with nitrosoguanidine and treated with FIG. 2. Schematic linkage map of Salmonella penicillin in minimal medium M-9 containing typhimurium (24).
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DAHP SYNTHASE MUTANTS OF SALMONELLA
in minimal medium. Their generation times of approximately 55 min were similar to that of wild-type LT2. The double mutant, strain SD401 (aroF3aroG9) with only the tryptophaninhibitable isoenzyme, had a generation time of approximately 240 min in minimal medium. Supplementation with 0.06 mM phenylalanine and tyrosine reduced the generation time to 75 min, and further addition of 0.01 mM tryptophan and 0.01 mM 4-aminobenzoate, 4-hydroxybenzoate, and 2,3-dihydrobenzoate failed to reduce the generation time of the double mutant to that of wild type. The effect of loss of one or more DAHP synthase isoenzymes on the remaining isoenzyme activities was investigated by growth of wild type and mutant strains on minimal and supplemented media (Table 3), In wild-type cells, the phenylalanine-inhibitable isoenzyme was the predominant one, whereas the tyrosineand tryptophan-inhibitable isoenzymes were present in much lower amounts. In cells grown on minimal medium, mutation of either aroF or aroH, structural genes for tyrosine- and tryptophan-inhibitable isoenzymes, respectively, resulted in only minor variations in the level of the remaining isoenzymic activities. However, mutation of aroG, structural gene for the phenylalanine-sensitive isoenzyme, resulted in a derepression of the tyrosine-inhibitable isoenzyme. A similar analysis of strains LT2, SD101 (aroF3), and SD201 (aroG9), grown on supplemented minimal medium, indicated that repression of the tyrosine-sensitive isoenzyme was independent of the other DAHP synthase activities. Derepressed levels of DAHP synthase (trp) in strain SD201 (aroG9) may be due to complete absence of the other two isoenzyme activities under these conditions. Growth of these strains on minimal medium supplemented only with tryptophan (0.1 to 0.5 mM) did not result in significant repression of the tryptophan-inhibitable isoenzyme (not shown). However, in strain SD401 (aroF3aroG9), grown on phenylalanine and tyrosine, 0.25 mM tryptophan repressed the tryptophan-inhibitable activity sixfold. DEAE-cellulose chromatography of DAHP synthase isoenzymes. Chromatography of wild-type extracts on DEAE-cellulose with a linear gradient of sodium chloride (29) failed to separate the DAHP synthase isoenzymes and resulted in 90% loss of overall activity. However, they were separated with a linear gradient of potassium phosphate buffer (pH 6.8) of 0.01 M to 0.50 M (Fig. 3). The largest activity, fractions 24-32, was completely inhibited by 1 mM
TABLE 3. Effect of growth with aromatic amino acids on DAHP synthase activities DAHP synthase
SD101 (aroF3) SD201 (aroG9) SD301
Mine Min + aaa Min Min + aaa Min Min + aaa Min
1.7 1.8 1.5 1.1 0.0 0.0 1.8
0.3 0.0 0.0 0.0 1.1 0.0 0.6
0.3 0.1 0.2 0.1 0.1 0.4 0.0
(aroHI) SD401 (aroF3 aroG9)
Min + 0.06 mM phe and tyr Min + 0.06 mM phe and tyr, and 0.1 mM trp Min + 0.15 mM phe and tyr, and 0.25 mM trp
a The reaction mixture (0.6 ml) contained 0.8 mM erythrose-4-P and 1.7 mM enolpyruvate-P (see text). Specific activity, micromoles per milligram of protein. bTotal activity minus activity measured in presence of 0.5 mM phenylalanine. cTotal activity minus activity measured in presence of 0.5 mM tyrosine. dTotal activity minus activity measured in presence of 0.5 mM phenylalanine and 0.5 mM tyrosine. e Min, Minimal medium; aaa, aromatic amino acids (0.06 mM phenylalanine [phe I and tyrosine [tyr] and 0.01 mM tryptophan [trp 1).
20 30 40 50 60 70 FRACTION NUMBER
FIG. 3. Chromatography of a wild-type extract on DEAE-cellulose with a linear gradient of phosphate buffer, pH 6.8.
DELEO AND SPRINSON
tyrosine, whereas activity in fractions 48-56 was completely inhibited by 1 mM phenylalanine. No cross-inhibition of either of these two activities by tyrosine or phenylalanine was observed at concentrations up to 1 mM. The enzyme activity in fractions 40-44 was not inhibited by tyrosine and/or phenylalanine and was considered to be the tryptophan-inhibitable DAHP synthase. Recovery of the phenylalanine-inhibitable isoenzyme was less than 1% of that found by assay of crude extracts. Addition of 1 mM dithiothreitol to the phosphate gradient increased recovery to approximately 10%. However, this isoenzyme was stabilized by 1 mM enolpyruvate-P, a tightly bound substrate of DAHP synthase enzymes (8, 27). Elution of DEAE-cellulose with a linear gradient of phosphate buffer containing 1 mM enolpyruvate-P (in the presence or absence of cell extract) indicated that enolpyruvate-P was retained on the column until the gradient was about 0.07 M phosphate, i.e., approximately the same concentration as was required to elute the tyrosinesensitive isoenzyme. Chromatography of an extract of wild-type cells in the presence of enolpyruvate-P (Fig. 4) showed that DAHP synthase in fractions 30-44 and fractions 60-80 was inhibited by tyrosine and phenylalanine, respectively, whereas the activity in fractions 48-54 was not inhibited by tyrosine and/or phenylalanine. The phenylalanine-inhibitable component now comprised the major isoenzyme activity as found by direct assay of cell extracts (Table 3). Chromatography in the presence of 1 mM enolpyruvate-P of an extract of strain SD101 (aroF3) resolved two peaks of DAHP synthase activity (Fig. 5). Enzyme activity in fractions 30-40 was not inhibited by tyrosine and/or phenylalanine, whereas the activity in fractions 52-70 was inhibited by phenylalanine. Minor tyrosine-inhibitable activity was both unstable and partially desensitized. An extract of strain SD301 (aroHl) was chromatographed in the presence of a linear gradient of 0 to 1.0 mM enolpyruvate-P, rather than a fixed 1 mM concentration, since in this extract tyrosineand phenylalanine-inhibitable isoenzymes were more effectively separated with a gradient of the substrate (Fig. 6). The DAHP synthase activity in fractions 20-30 and 46-60 was inhibited by tyrosine and phenylalanine, respectively. Chromatography of an extract of strain SD201 (aroG9) in the presence of 1 mM enolpyruvate-P resulted in poor resolution of the tyrosine- and tryptophan-inhibitable isoenzyme, and this extract was chromatographed in the absence of
FIG. 4. Chromatography of a wild-type extract on DEAE-cellulose with a linear gradient of phosphate buffer containing I mM enolpyruvate-P. Inset, elution pattern of enolpyruvate-P. The phosphate buffer gradient curve was similar to that of Fig. 3 and was omitted in Fig. 4 to 8.
z 0 cr a-
FIG. 5. Chromatography of an extract of strain SD1OJ (aroF3) on DEAE-cellulose with a linear gradient of phosphate buffer containing 1 mM enolpyruvate-P.
substrate (Fig. 7). Synthase activity in fractions 40-50 was inhibited by tyrosine, whereas the activity in fractions 60-70 was not inhibited by tyrosine and/or phenylalanine. Chromatography in the presence of 1 mM enolpyruvate-P of an extract of strain SD401 (aroF3aroG9) gave only one major DAHP synthase fraction which was not inhibited by tyrosine and/or phenylalanine (Fig. 8). A minor fraction of an unstable, partially desensitized, tyrosine-inhibitable isoenzyme was observed similar to that obtained in chromatography of an extract of strain SD101, the parent strain of the double mutant.
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DAHP SYNTHASE MUTANTS OF SALMONELLA
tion to tryptophan, resulted in lowering the expected absorbance (Table 4), and lower levels -20 E 3of erythrose-4-P minimized interference in the assay. This result was observed with erythrose-4-P prepared by lead tetraacetrate oxidation of glucose-6-P (3), which is contami2nated with unreacted starting material and various side products (e.g., glyceralde~~~~~~~~~~~~~-0.6~ hyde-3-P), or with erythrose-4-P obtained by %00, ~~~0.4 hydrolysis of its dimethyl acetal cyclohexylam?cbakl"O monium salt. Since erythrose-4-P lowered the optical density of the assay only when present with trypto20 30 40 50 60 70 10 phan during periodate oxidation of DAHP (not FRACTION NUMBER shown), some expected periodate oxidation FIG. 6. Chromatography of an extract of strain products of erythrose-4-P, and related. comSD301 (aroHI) on DEAE-cellulose with a linear pounds, were tested for their effect on the assay gradient phosphate buffer and a linear gradient of 0 to (Table 5). Glyoxal and glycoaldehyde-2-P, in 7E
, IS,2 aZX 0.8
1.0 mM enolpyruvate-P.
3 0 0
20 30 40 50 60 FRACTION NUMBER
FIG. 7. Chromatography of an extract of strain SD201 (aroG9) on DEAE-cellulose with a linear gradient of phosphate buffer.
Interference by L-tryptophan in the DAHP synthase assay. The availability of stable, partially purified preparations of DAHP synthase activity from strain SD401 made possible a direct investigation of inhibition of this enzyme by tryptophan. Previously, aroH-specified isoenzyme was measured indirectly as phenylalanine- and tyrosine-uninhibitable activity, owing to lowering by tryptophan of color obtained in the periodate-thiobarbiturate assay for DAHP (9, 17). Initial experiments indicated that tryptophan alone at higher than 2.5 mM reacted in the assay and gave considerable blank absorptions. Tryptophan (0.5 to 2.5 mM), together with synthetic DAHP, did not decrease the expected absorbance of the colorimetric assay, but increased it in most determinations. However, the presence of erythrose-4-P, in addi-
FIG. 8. Chromatography of an extract of strain SD401 (aroF3aroG9) on DEAE-cellulose with a linear gradient of phosphate buffer containing 1 mM enolpyruvate-P.
TABLE 4. Effect of tryptophan and erythrose-4-P on assay of synthetic DAHP"
0.10 0.20 0.30 0.10 0.20 0.30
0.15 0.15 0.15
0.289 0.322 0.303 0.286 0.273 0.288 0.252 0.241
Change (%) +11 + 4 - 1 - 6 0 -13 -17
Except where otherwise noted, the assay mixture (0.2 ml) contained 0.0165 ;imol of DAHP. (The blank solution consisted of all reagents except for DAHP.) b Assays were done in duplicate and had a standard deviation of less than 4%. OD,4,, Optical density at a
549 nm. c This amount of tryptophan corresponds to a 1 mM concentration in the enzyme assays.
DELEO AND SPRINSON
TABLE 5. Effect of periodate oxidation products of erythrose-4-P and of related compounds on assay of synthetic DAHP in presence of tryptophana Compound added
Glyoxal Glyoxylic acid Formaldehyde Glycoaldehyde 2_Pb
0.10 0.20 0.10 0.20 0.10 0.20 0.10 0.20 0.10 0.20 0.30
0.388 0.328 0.294 0.344 0.324 0.383 0.359 0.391 0.378 0.276 0.271 0.284
-15 -26 -12 -17 -1 -8 0 -3
significant in view of recent findings on the regulation of tyrosine biosynthesis. Operator mutants (tyrOc) of Salmonella have been isolated and shown to be constitutively and coordinately derepressed for the tyrosine biosynthetic enzymes, DAHP synthase (tyr) and prephenate dehydrogenase, which are specified by aroF and tyrA, respectively (16). A second tyrosine regulatory mutation, unlinked to either tyrA or aroF and designated tyrR, also results in constitutive and coordinate derepression of the TABLE 6. Effect of tryptophan analogues on assay of DAHPa
-29 -30 - 27
A solution (0.2 ml) containing 0.0165 Amol of DAHP and 0.15 Amol of L-tryptophan was assayed. OD549, Optical density at 549 nm. bThis compound had no effect on the assay in the absence of tryptophan.
addition to erythrose-4-P, depressed the optical density of the assay in the presence of tryptophan, whereas in its absence these compounds had no effect. The results of these experiments suggested that the products of an interaction between tryptophan and an aldehyde was interfering in the periodate-thiobarbiturate assay of DAHP. Hence, the effect of structural changes in the tryptophan molecule was tested in the assay. 1-Methyl and 2-methyl tryptophan did not interfere significantly, whereas the other derivatives resembled tryptophan in their effect on the assay (Table 6). The effect of L-tryptophan, 1-methyl-DLtryptophan, and 2-methyl-DL-tryptophan on the activity of aroH specified isoenzyme was studied under conditions of minimum interference with the assay (Table 7). At 1 mM concentrations, L-tryptophan and 2-methyl-DL-tryptophan inhibited enzyme activity about 30%, whereas 1-methyl-DL-tryptophan was not inhibitory. Higher concentrations did not increase inhibition. DISCUSSION Biochemical and genetic evidence, described above, indicates that the initial reaction of aromatic amino acid biosynthesis in Salmonella is catalyzed by three isoenzymes under control of three unlinked genes. While this work was in progress, similar studies were reported in E. coli (4, 29). The three structural genes of DAHP synthase in Salmonella were mapped by assuming similar locations to those found in E. coli. The linkage of aroF and tyrA (Table 2) is
0.337 0.336 0.329 0.280 0.326 0.316 0.294 0.327 0.311 0.230 0.335 0.346 0.341 0.332 0.334 0.339
0 0.10 0.20 0 0.10 0.20 0 0.10 0.20 0 0.10 0.20 0 0.10 0.20
0 -3 -17 -3 -6 -13 -3 -9 - 32 -1 +3 +1 -2 -1 0
The reaction mixture (0.2 ml) contained 0.0165
,4mol of DAHP. OD649, Optical density at 549 nm. b0.15 Amol. C
TABLE 7. Inhibition of DAHP synthase (trp) by tryptophan and tryptophan analoguesa Synthetic Inhibitor
None L-Tryptophan 1-Methyl-DL-tryptophan 2-Methyl-DL-tryptophan
Enzymic assay mixture"
0.205 0.185 0.207
0.302 0.179 0.324
a DAHP synthase (trp) was obtained from chromatographic fractions of extracts of strain SD401 (aroF3aroG9). Parallel assays were carried out with 0.07 mM synthetic DAHP to evaluate interference with assay. Concentrations of tryptophan and its analogues were 1 mM. OD6,4, Optical density at 549 nm. b See Table 3, footnote a.
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DAHP SY'1THASE MUTANTS OF SALMONELLA
aroF and tyrA gene products (15). These results have been interpreted as evidence for a unit of regulation in tyrosine biosynthesis in Salmonella. Similar results were obtained in E. coli (21, 30). Linkage of structural genes of tryptophan-inhibitable DAHP synthase and dehydroquinase has yet to be extensively studied in relation to control of the aromatic pathway. However, preliminary experiments with strains aroF3 and aroG9 grown on limiting tryptophan indicated coordinate derepression of the aroH and aroE genes (E. G. Gollub, personal communication). Loss of a DAHP synthase isoenzyme activity did not significantly alter the level of the remaining two isoenzymes, except in strain SD201 which lacked the phenylalanine-inhibitable isoenzyme and was derepressed for DAHP synthase (tyr). The tyrosine-inhibitable isoenzyme displayed the clearest pattern of endproduct control. It was partially repressed in wild type on minimal medium but completely repressed in wild-type cells and strain SD201 grown on minimal medium plus aromatic amino acids. Both phenylalanine- and tyrosine-inhibitable DAHP synthase isoenzymes in column fractions were completely inhibited by 1 mM end product. There was no cross-inhibition of the three isoenzymes by either L-phenylalanine or L-tyrosine. Under our growth conditions for the double mutant SD401, DAHP synthase (trp) was repressed by high concentrations of tryptophan. This isoenzyme was inhibited only about 30% by 1 mM concentrations of tryptophan. The three DAHP synthase isoenzymes were separated on DEAE-cellulose with a phosphate gradient, but it was necessary to stabilize the phenylalanine-inhibitable DAHP synthase with 1 mM enolpyruvate-P in the eluting gradient. Stabilization of DAHP synthase isoenzymes by enolpyruvate-P during chromatography and against heat denaturation has been reported previously (25, 27) and is in accord with evidence for an ordered sequential mechanism in DAHP synthase (tyr) with obligatory binding of enolpyruvate-P to the enzyme prior to binding of the second substrate, erythrose-4-P (8). Interference by tryptophan in the thiobarbituric acid assay for DAHP appeared to require the presence of erythrose-4-P during periodate oxidation. Erythrose-4-P acted directly or after cleavage by periodate to other aldehydic compounds, e.g., glycolaldehyde-2-P. Furthermore, a methyl group on N-1 or C-2 of tryptophan prevented interference in the assay, whereas 2,3-dihydrotryptophan and a-methyl trypto-
phan were still effective. The facile condensation of C-2 of indoles with aldehydes may be responsible for the observed effects. Although 0.016 umol of glycolaldehyde-2-P is presumably formed by periodate cleavage of DAHP, the amount of aldehyde added (Table 5) was 6to 18-fold higher. Glyoxal may exert its effect as a result of being present in polymeric form (12, 23) and, thus, unavailable to rapid cleavage by periodate. The previously reported interference by erythrose-4-P and diols in the DAHP assay as a result of competition for periodate (10) probably does not occur in our procedure, which is conducted with a much higher concentration of periodate (125-fold as compared to 10-fold). Interference in the assay could be minimized by using lower concentrations of erythrose-4-P. Under these conditions, 1 mM L-tryptophan and 2-methyl-DL-tryptophan gave inhibitions of 30% of DAHP synthase activity that was not inhibited by phenylalanine or tyrosine, whereas 1-methyl tryptophan was not inhibitory. The observation that aroH-specified isoenzyme is only partially inhibited is in agreement with recent studies in E. coli (5, 14). In contrast, the tryptophan-sensitive isoenzyme of Claviceps paspali was 95% inhibited by 0.125 mM L-tryptophan (11), whereas the DAHP synthase of Streptomyces aureofaciens (18) was 65% inhibited by 0.01 mM L-tryptophan. Both enzymes also were inhibited by 2-methyl tryptophan but not by 1-methyl tryptophan. ACKNOWLEDGMENTS This work was supported by grants from the American Cancer Society, the American Heart Association, the Public Health Service (AM00546-23 from the National Institute of Arthritis, Metabolism and Digestive Diseases), and the National Science Foundation. A.B.D. was supported by Public Health Service training grant GM255 and predoctoral fellowship F01 GM38252 from the National Institute of General Medical Sciences. D.B.S. is a career investigator of the American Heart Association. We thank Edith G. Gollub for the many helpful discussions. LITERATURE CITED 1. Adams, M. H. 1959. Bacteriophages. Interscience, New
York. 2. Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965. Optimal conditions for mutagenesis by N-methyl-N'nitro-N-nitroso-guanidine in Escherichia coli K-12. Biochem. Biophys. Res. Commun. 18:788-795 3. Ballou, C. E., and D. L. MacDonald. 1963. Synthesis of
aldose phosphates by glycol cleavage, p. 293-297. In R. L. Whistler and M. L. Wolfrom (ed.), Methods in carbohydrate chemistry, vol. II. Academic Press Inc., New York. 4. Brown, K. D. 1968. Regulation of aromatic amino acid biosynthesis in Escherichia coli K12. Genetics 60:31-48. 5. Camakaris, J., and J. Pittard. 1974. Purification and
DELEO AND SPRINSON
properties of 3-deoxy-D-arabinoheptulosonic acid7-phosphate synthetase (trp) from Escherichia coli. J. Bacteriol. 120:590-597. 6. Clark, V. M., and A. J. Kirby. 1966. Phosphoenol pyruvic acid (monocyclohexylammonium salt). Biochem. Prep. 11:101-104. 7. Dayan, J., and D. B. Sprinson. 1971. Enzyme alterations in tyrosine and phenylalanine auxotrophs of Salmonella typhimurium. J. Bacteriol. 108:1174-1180. 8. DeLeo, A. B., J. Dayan, and D. B. Sprinson. 1973. Purification and kinetics of tyrosine-sensitive 3-deoxyD-arabino-heptulosonic acid 7-phosphate synthetase from Salmonella. J. Biol. Chem. 248:2344-2353. 9. Doy, C. H. 1967. Tryptophan as an inhibitor of 3-deoxyarabino-heptulosonate 7-phosphate synthetase. Biochem. Biophys. Res. Commun. 26:187-192. 10. Eberspacher, J., and F. Lingens. 1970. Untersuchungen zur Stbrung des Phospho-2-oxo-3-desoxyheptonatAldolase-Tests. Hoppe-Seyler's Z. Physiol. Chem. 351:373-376. 11. Eberspacher, J., H. Uesseler, and F. Lingens. 1970. Eigenschaften der Phospho-2-oxo-3-desoxyheptonataldolase aus Claviceps SD 58. Hoppe-Seyler's Z. Physiol. Chem. 351:1465-1474. 12. Fischer, H. 0. L., and C. Taube. 1926. Uber Glyoxal. Ber. Chem. Ges. 59:851-856. 13. Friedemann, T. E., and G. E. Haugen. 1943. Pyruvic acid. II. The determination of keto acids in blood and urine. J. Biol. Chem. 147:415-442. 14. Gibson, F., and J. Pittard. 1968. Pathways of biosynthesis of aromatic amino acids and vitamins and their control in microorganisms. Bacteriol. Rev. 32:465-492. 15. Gollub, E. G., K. P. Liu, and D. B. Sprinson. 1973. tyrR, a regulatory gene of tyrosine biosynthesis in Salmonella typhimurium. J. Bacteriol. 115:1094-1102. 16. Gollub, E. G., and D. B. Sprinson. 1969. A regulatory mutation in tyrosine biosynthesis. Biochem. Biophys. Res. Commun. 35:389-395. 17. Gollub, E. G., H. Zalkin, and D. B. Sprinson. 1967. Correlation of genes and enzymes, and studies on regulation of the aromatic pathway in Salmonella. J. Biol. Chem. 242:5323-5328. 18. Gorisch, H., and F. Lingens. 1971. 3-Deoxy-D-arabinoheptulosonate-7-phosphate synthase of Streptomyces
J. BACTERIOL. aureofaciens tu 24. II. Repression and inhibition by tryptophan and tryptophan analogues. Biochim. Biophys. Acta 242:630-636. 19. Kornberg, A., and W. E. Pricer, Jr. 1951. Enzymatic phosphorylation of adenosine and 2,6-diaminopurine riboside. J. Biol. Chem. 193:481-495.
20. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 21. Mattern, E. E., and J. Pittard. 1971. Regulation of tyrosine biosynthesis in Escherichia coli K-12: Isolation and characterization of operator mutants. J. Bacteriol. 107:8-15. 22. Patte, J. C., and G. N. Cohen. 1965. Isolement et proprietes d'un mutant d'Escherichia coli depourvu d'aspartokinase sensible a la lysine. Biochim. Biophys. Acta 99:561-563. 23. Raudnitz, H. 1944. The constitution of trimeric glyoxal. Chem. Ind. 63:327; 366. 24. Sanderson, D. E. 1972. Linkage map of Salmonella typhimurium, edition IV. Bacteriol. Rev. 36:558-586. 25. Simpson, R. J., B. E. Davidson, T. A. A. Dopheide, S. Andrews, and J. Pittard. 1971. Purification and properties of 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase (phe) from a XaroG+ transductant of Escherichia coli. J. Bacteriol. 107:798-805. 26. Smith, L. C., J. M. Ravel, S. R. Lax, and W. Shive. 1962. The control of 3-deoxy-D-arabino-heptulosonic acid
7-phosphate synthesis by phenylalanine and tyrosine. J. Biol. Chem. 237:3566-3570. Staub, M., and G. Denes. 1969. Purification and properties of the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (phenylalanine sensitive) of Escherichia coli K12. Biochim. Biophys. Acta 178:588-598. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 218:97-106. Wallace, B. J., and J. Pittard. 1967. Genetic and biochemical analysis of the isoenzymes concerned in the first reaction of aromatic biosvnthesis in Escherichia coli. J. Bacteriol. 93:237-244. Wallace, B. J., and J. Pittard. 1969. Regulator gene controlling enzymes concerned in tyrosine biosynthesis in Escherichia coli. J. Bacteriol. 97:1234-1241.