May 18, 1973 - IRA SCHILDKRAUT AND SHELDON GREER. Departments of .... min with 210 g of' glass beads of' 100 to 200 um diameter. The lysate was ...
Vol. 115, No. 3 Printed in U.S.A.
JOURNAL OF BACrERIOLOGY, Sept. 1973, p. 777-785 Copyright 0 1973 American Society for Microbiology
Threonine Synthetase-Catalyzed Conversion of Phosphohomoserine to a-Ketobutyrate in Bacillus subtilis IRA SCHILDKRAUT AND SHELDON GREER
Departments of Microbiology and Biochemistry, University of Miami, Coral Gables, Florida 33146 Received for publication 18 May 1973
An enzyme activity of Bacillus subtilis has been found that catalyzes the dephosphorylation and deamination of phosphohomoserine to a-ketobutyrate, resulting in a bypass of threonine in isoleucine biosynthesis. In crude extracts of a strain deficient in the biosynthetic isoleucine-inhibitable threonine dehydratase, phosphohomoserine was converted to a-ketobutyrate. Phosphohomoserine conversion to a-ketobutyrate was shown not to involve a threonine intermediate. Single mutational events affecting threonine synthetase also affected the phosphohomoserine-deaminating activity, suggesting that the deamination of phosphohomoserine was catalyzed by the threonine synthetase enzyme. It was demonstrated in vivo, in a strain deficient in the biosynthetic threonine dehydratase, that isoleucine was synthesized from homoserine without intermediate formation of threonine.
Previous work in this laboratory (17) demonstrated that threonine synthetase (EC 2.4.99.2; phosphohomoserine _ threonine + inorganic phosphate) of Bacillus subtilis can also catalyze the deamination of threonine to yield a-ketobutyrate. This result, and consideration of the catalytic mechanisms of threonine synthetase of Neurospora (7, 8) and the biodegradative threonine dehydratase (EC 4.2.1.16; threonine -_ a-ketobutyrate + NH8) of Escherichia coli (15), led us to predict that threonine synthetase of B. subtilis can catalyze the dephosphorylation and deamination of phosphohomoserine to aketobutyrate without intermediate formation of threonine. The synthesis of threonine from phosphohomoserine proceeds by formation of an a,, unsaturated intermediate, a-aminocrotonate, to which water is added forming the pyridoxal Schiff base of threonine (7, 8). a-Aminocrotonate is also an intermediate in the conversion of threonine to a-ketobutyrate catalyzed by threonine dehydratase (15). However, in the threonine dehydratase reaction, a-aminocrotonate is eliminated from the enzyme and is spontaneously hydrolyzed to a-ketobutyrate and ammonia. It is proposed that if a-aminocrotonate were eliminated from the threonine synthetase enzyme, direct synthesis of a-ketobutyrate from
phosphohomoserine could occur without intermediate formation of threonine. This paper presents evidence for an enzymatic activity associated with threonine synthetase that catalyzes the dephosphorylation and deamination of phosphohomoserine. In addition, evidence is provided for the in vivo conversion of homoserine to isoleucine without intermediate formation of threonine, the usually recognized intermediate in isoleucine biosynthesis. (A summary of this paper was presented at the 73rd Annual Meeting of the American Society for Microbiology, 6-11, May 1973.) MATERIALS AND METHODS Bacterial-strains. Mutant strains of B. subtilis (Table 1) were derived from strains 23 and 168 indoleof Burckholder and Giles (3) spontaneously or by in vitro mutagenesis of deoxyribonucleic acid followed by transformation (20). All mutant strains utilized in this study were deficient in the biosynthetic, isoleucine-inhibitable threonine dehydratase (17, 20, 21). The phenotypic expression of the ile mutation is isoleucine auxotrophy. The residual threonine dehydratase activity observed in these strains is known to be an associated activity of threonine synthetase (17). Many of the strains used possessed either or both of two mutations that partially suppress the original ile mutation. One suppressor, sprA, results in a 5- to
777
SCHILDKRAUT AND GREER
778
TABLE 1. Isoleucine and threonine auxotrophic mutations and mutations of partial reversion to
prototrophy Strain
ile-3 ile-3, sprA-44 ile-3, sprB-24
ile-3, sprA-8, sprB-24 ile-3, sprA-44, thrB-37 ile-3, sprA-44, tdm-3 ile-3, sprA-44, thrA-47
Amino acid requirement
Isoleucine Isoleucine or homoserine or threonine Isoleucine or homoserine or threonine None Isoleucine plus threonine Isoleucine Threonine
10-f'old derepression of the threonine biosynthetic enzymes and therefore a 5- to 10-fold derepression of' the threonine synthetase-associated threonine dehydratase (17). The other suppressor, sprB, maps within the gene encoding threonine synthetase, and results in a 90% loss of' threonine synthetase activity, but no apparent change in the threonine synthetaseassociated threonine dehydratase activity (17). Either suppressor mutation alone allows for growth of a strain on threonine or homoserine as well as isoleucine (20). Together, the two suppressor mutations result in prototrophy (20). The thrB mutation results in a loss of threonine synthetase and threonine synthetaseassociated threonine dehydratase activity (17), tdm causes a 90% decrease in threonine synthetase activity and a lack of threonine synthetase-associated threonine dehydratase activity (17), and thrA is a mutation that results in a lack of' homoserine kinase activity
(17).
Chemicals. Amino acids were obtained from Sigma Chemical Co. and Calbiochem. Alcohol dehydrogenase, lactate dehydrogenase, deoxyribonuclease, and ribonuclease were obtained from Sigma Chemical Co. Lysozyme (salt-free) was a product of Worthington Biochemical Corp. Uniformly labeled '4C-L-threonine, '4C -L-methionine, and '4C-L-isoleucine, generally labeled 3H-L-threonine, and 4- '4C-D, L-homoserine were obtained from Schwarz/Mann. Uniformly labeled '4C-L-homoserine was obtained from Amersham/Searle. Synthesis of phosphohomoserine. Phosphohomoserine was synthesized from homoserine by use of' the homoserine kinase obtained from baker's yeast, by a modification of the method of' Flavin (6). Baker's yeast was obtained commercially and was washed in 0.1 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 8.0. Disruption of yeast cells was accomplished by treating 70 g (wet weight) in 90 ml of' 0.1 M Tris-hydrochloride (pH 8.0) at 0 C in a Sorvall Omnimixer at one-half' maximum speed for 20 min with 210 g of' glass beads of' 100 to 200 um diameter. The lysate was centrif'uged at 27,000 x g for 15 min at 0 C, and the supernatant f'luid was recentrif'uged at 43,000 x g at 0 C for 30 min. An ammonium sulfate fraction was obtained between 30 and 43% saturation. This fraction was washed in 45% saturated ammonium sulfate in 0.1 M Tris-hydrochloride (pH 8.0) and was finally dissolved in 30 ml of 40
J. BACTERIOL.
mM Tris-hydrochloride (pH 7.5) containing 4 mM reduced glutathione. The reaction mixture consisted of' 10 mmol of' Tris-hydrochloride (pH 7.5), 5 mmol of L-homoserine, 500 kmol of MgSO4, 3 mmol of adenosine triphosphate (ATP), and 1 mmol of NaF in a total volume of' 100 ml. The pH was adjusted to 7.5 with KOH, and 8 ml of the yeast 30 to 43% ammonium sulfate fraction was added to the reaction mixture. The reaction was monitored, and phosphohomoserine was purified according to a modification (17) of the method of' Wormser and Pardee (22). The compound that was synthesized and purified as described above was demonstrated by the following criteria to be phosphohomoserine. The compound migrated with an R, value described for phosphohomoserine (17) in descending chromatography with Whatman no. 52 paper and 88% phenol as a solvent. The compound gave coincident positive ninhydrin and phosphate reactions. When the compound was chromatographed in 100-fold excess, no additional ninhydrin spots were found, indicating that the compound had less than 1% contaminating amino acids. A quantitative ninhydrin (14) and quantitative bound phosphate determination (5, 10) gave a phosphate to amino acid molar ratio of' 0.94. The phosphate determination demonstrated that less than 1% inorganic phosphate was present. Incubation of the compound in crude extracts of B. subtilis resulted in the production of threonine. Growth of cells and preparation of cell extract. Cells were grown in Spizizen's minimal medium (1) supplemented with 0.5% glucose and 100 Ag of' the amino acid(s)/ml required by the particular strain. Cells were harvested in mid-log phase and washed once with the buffer to be used in subsequent enzyme assays. The pellet was then resuspended in 10 to 20 volumes of buffer, and the cell suspension was used immediately or frozen in portions of 1 to 2 ml at -30 C. The specific activities of' the enzymes studied were the same in fresh and frozen cells. Cells were lysed with 200 Mg of lysozyme/mi at 37 C. Protein concentration was determined by the method of Lowry et al. (13) with bovine serum albumin as standard. Enzymatic formation of a-ketobutyrate. The method used was a modification of that described by Umbarger and Brown (19). The assay was carried out in a 0.5-ml volume containing 100 mM potassium phosphate (pH 8.0), 0.1 mM pyridoxal phosphate, substrate (threonine, homoserine, or phosphohomoserine), and 0.2 to 1.0 mg of crude extract protein. The reaction mixture was incubated at 37 C for the indicated times and was assayed for keto acids colorimetrically by Greenberg's modif'ication (11) of the method described by Friedemann and Haugen (9). Monosodium a-ketobutyrate was the standard. Control tubes contained no substrate. The initial rate of a-ketobutyrate formation was proportional to protein concentration. Threonine synthetase. The method used was a modif'ication of' that described by Flavin (6). The assay was carried out in a 1.25-mi volume containing 0.6 mM phosphohomoserine, 0.08 mM pyridoxal phosphate, 80 mM potassium phosphate (pH 8.0),
VOL. 115, 1973
PHOSPHOHOMOSERINE CONVERSION TO a-KETOBUTYRATE
and crude extract. Control tubes contained no phosphohomoserine. The reaction was stopped by boiling for 1 min, and the protein precipitate was removed by centrifugation at 0 to 5 C. Threonine was assayed by periodate oxidation to acetaldehyde, which was measured by the amount of reduced nicotinamide adenine dinucleotide (NADH) oxidized in the presence of alcohol dehydrogenase (6). Controls for the periodate oxidation consisted of homoserine, phosphohomoserine, and a-ketobutyrate, each of which resulted in less than 1% oxidation of NADH as compared with the threonine standard. Fate of "C-D, u.homoserine and 3H-ithreonine. The radioactive metabolites present at 0, 40, and 240 min in a reaction mixture for enzymatic aketobutyrate formation containing 3H-L-threonine, "C-D, L-homoserine, and ATP were analyzed by descending chromatography. Samples of the reaction mixture (10 ,liters) were applied to Whatman no. 52 (acid-washed) filter paper, and 88% phenol was utilized as the solvent. Amino acid and a-ketobutyrate standards were chromatographed in parallel with the samples on each chromatogram, and the amino acid standards were identified by ninhydrin reagent spray. Phosphohomoserine, in addition to a positive ninhydrin test, gave a positive reaction in the phosphate test. a-Ketobutyrate was demonstrated with bromocresol green spray (2). As an additional control, '4C-D, L-homoserine and 3H-L-threonine were chromatographed in the presence of unlabeled reaction mixture. The chromatograms were dried at room temperature and cut into fractions. Each fraction was eluted with water and counted in a liquid scintillation spectrometer with correction made for spillover between 14C and 3H channels. Conversion of homoserine to isoleucine in vivo. Strain ile-3, sprB-24 was grown in unlabeled medium to mid-log phase and then inoculated into 5 ml of identical medium containing "4C-labeled amino acid. At the end of log growth, which corresponded to approximately two generations in labeled medium, the culture was centrifuged and washed in 0.1 M potassium phosphate buffer (pH 8.0), lysed with 200 ug of lysozyme/ml, and treated with 500 ,g of ribonuclease/ml and 30 gg of deoxyribonuclease/ml. The protein was precipitated and washed three times with 100% saturated ammonium sulfate at 0 C. The pellet was dissolved in 5.0 ml of water. A 2-ml sample was dialyzed overnight against two changes of 3 liters of water. The protein was hydrolyzed in 6 N HCl by heating to 110 C for 12 h. The hydrolysate was dried and redissolved in 0.1 ml of water; 10 Mliters was applied to Whatman 3 MM paper, dried, and chromatographed with butanol-acetic acid-water, 12:5:3. The paper was dried, and the distribution of radioactivity was determined with a Baird Atomic radiochromatogram scanner. "4C-labeled standards were run through the hydrolysis procedure and used as controls in the chromatographic system. The relative amounts of radioactivity were determined by integrating the areas under the peaks of the radioactivity scan. Counts per minute per radioactive area were obtained by cutting out radioactive areas and counting by liquid scintillation spectroscopy.
779
RESULTS Conversion of homoserine and phosphohomoserine to a-ketobutyrate. In crude extracts of strains lacking the biosynthetic isoleucine end product-inhibitable threonine dehydratase, a-ketobutyrate formation from homoserine or phosphohomoserine occurred at a rate similar to that obtained with threonine (Fig. 1). The formation of a-ketobutyrate from phosphohomoserine was linear with time and protein concentration (Fig. 2). Since all strains utilized in this study lacked the biosynthetic isoleucineinhibitable threonine dehydratase, the only remaining threonine dehydratase activity was the threonine synthetase-associated threonine dehydratase (17). In the case of homoserine, ATP was added to the reaction mixture so that homoserine could be converted to phosphohomoserine by homoserine kinase (EC 2.7.1.39). The conversion of homoserine to a-ketobutyrate did not occur when ATP was not added to the reaction mixture, although a-ketobutyrate was formed from phosphohomoserine (Fig. 3). Furthermore, a mutant strain lacking homoserine 0.4-
~~~~50
NOMISERINE
(+ATPJ
~0.3-
c-
*N TNREONINE
__
m
0.2 m
Lhi
.5~ ~ ~v
mM
PNOSPNONSMOSERINE
MINUTES FIG. 1. Conversion of homoserine, phosphohomoserine, and threonine to a-ketobutyrate. Homoserine, 50 mM, plus 10 mM ATP and 5 mM MgSO4 (A), phosphohomoserine, 5 mM (0), or threonine, 50 mM (0), was incubated in the presence of crude extract of ile-3, sprA-8, sprB-24. The formation of a-ketobutyrate was determined colorimetri-
cally.
J. BACTERIOL.
SCHILDKRAUT AND GREER
780
0.8/
W
-0.6/ / -/*
Y 0.4-
*./ /
~
/
2
0 /° * /o/D
- 0.2-
/ o/: , /
120 180 MINUTES FIG. 2. Formation of a-ketobutyrate as a function of time and protein concentration. Phosphohomoser0
60
standard on Whatman no. 52 chromatography paper with 88% phenol as solvent (Table 2). Conversion of phosphohomoserine to a-ketobutyrate without intermediate threonine formation. The conversion of phosphohomoserine to a-ketobutyrate could occur via a twostep catalytic process, threonine synthetase and the threonine synthetase-associated threonine dehydratase, or by the alternatively proposed direct dephosphorylation and deamination of phosphohomoserine. To eliminate the possibility that phosphohomoserine is converted to a-ketobutyrate via a threonine intermediate, we assayed the formation of a-ketobutyrate from low concentrations of phosphohomoserine and threonine in crude extract. The intention of lowering the substrate concentration was to demonstrate a difference in the Km for aketobutyrate formation from phosphohomoserimne and threonine. Since the Km for threonine of the threonine synthetase-associated threonine dehydratase activity is large, approximately 50 mM (17), a low concentration of phosphohomoserine might be converted to a-ketobutyrate at a faster rate than a low concentration of threonine. The rate of a-ketobutyrate formation from 2.5 mM phosphohomoserine was at least 20-fold
ine, 5 mM, was incubated in the presence of crude extract of ile-3, sprA-8, sprB-24. The formation of a-ketobutyrate was determined colorimetrically. Reaction vessels contained 1.6 mg (0), 0.80 mg (0), or 0.40 mg (0) of protein per ml of reaction volume.
kinase was unable to convert homoserine to a-ketobutyrate, indicating that phosphohomoserine is a necessary intermediate for the formation of a-ketobutyrate from homoserine (Fig. 4). The colorimetric assay for a-ketobutyrate measured any keto acid. It was, therefore, necessary to show that the keto acid formed enzymatically from homoserine and phosphohomoserine was a-ketobutyrate. A sample of a reaction mixture in which keto acid was produced from homoserine in the presence of crude extract, and ATP was incubated with lactate dehydrogenase and NADH. The rate of oxidation of NADH to NAD with the keto acid formed enzymatically from homoserine was similar to that observed with a-ketobutyrate, rather than with pyruvate (Fig. 5). This indicates that the keto acid formed is not pyruvate and is consistent with the formation of aketobutyrate. Furthermore, 14C -D, L-homoserine incubated with crude extract and ATP was converted to a "4C-labeled product that migrated coincident with an a-ketobutyrate
0.152.5 mM
PNISPNONOSMSEUINE /
,
0.10l /
-
_
* 0.05 ° M
Es ImIUNSEIINE V-ATPJ 16
23
30
46 5I MINUTES
6I 7ii
9 0Isio
FIG. 3. Lack of conversion of homoserine to aketobutyrate when ATP is excluded from the reaction
mixture. Homoserine, 5 mM (0), or phosphohomoser-
ine, 2.5 mM (0), was incubated in the presence of crude extract of ile-3, sprA-8, sprB-24. The formation of a-ketobutyrate was determined colorimetrically.
/~ ~ ~
PHOSPHOHOMOSERINE CONVERSION TO a-KETOBUTYRATE
VOL. 115, 1973
0.2
5
0.1
mu PHOSPIOHIMOSCRINE
/
5 mN NONOSERINE (+ATPJ
O
Q 15
0
30 MINUTES
0
45
60
FIG. 4. Lack of conversion of homoserine to aketobutyrate in a strain lacking homoserine kinase. Homoserine, 5 mM, plus 10 mM ATP and 5 mM MgS04 (0), or phosphohomoserine, 5 mM (0), was incubated in the presence of crude extract of ile-3, sprA-44, thrA-47. The formation of a-ketobutyrate was determined colorimetrically.
higher than the rate from 2.5 mM threonine (Fig. 6). The rate of conversion of 25 mM phosphohomoserine was twofold higher than the rate from 25 mM threonine. At a low concentration of threonine, the rate of a-ketobutyrate formation was substantially lower than at a high threonine concentration (Fig. 6), as was expected because of the high Km of the threonine synthetase-associated threonine dehydratase for threonine. Since a low concentration of phosphohomoserine was converted to a-ketobutyrate more readily than was threonine, the synthesis of a-ketobutyrate from phosphohomoserine may occur without intermediate threonine formation. This is consistent with the suggestion that there is direct conversion of phosphohomoserine to a-ketobutyrate. It is conceivable, however, that phosphohomoserine activates the threonine synthetase-associated threonine dehydratase activity by lowering the Km for threonine. If this were the case, low concentrations of threonine could be efficiently converted to a-ketobutyrate in the presence of phosphohomoserine. To rule out this possibility, we performed an experiment in which both homoserine and threonine were present in the same incubation
781
mixture. The fate of "4C-D,L-homoserine and 3H-L-threonine was determined by chromatography of samples of the reaction mixture after various times of incubation (Table 2). The concentrations of substrates were chosen so that conversion of threonine to a-ketobutyrate via the threonine synthetase-associated threonine dehydratase activity would occur at a reduced rate, unless the threonine synthetase-associated threonine dehydratase activity was activated by phosphohomoserine. There was a rapid conversion of "C-homoserine to 04C-phosphohomoserine. Some "4C-phosphohomoserine is found in the zero-time reaction mixture, probably as a result of the time it takes to apply the reaction mixture and dry it on the paper. At later times, the 04C-phosphohomoserine is converted to 14C_ threonine and "4C-a-ketobutyrate in an approximate molar ratio of 2: 1. It should be noted that approximately one-half of the starting homoserine remains unaltered during the incubation; this corresponds to the D isomer of homoserine. In contrast to the conversion of phosphohomoserine to a-ketobutyrate, the rate of formation of a-ketobutyrate from 3H-threonine was less than 3% of the rate from phosphohomoserine. This experiment rules out the possibility of phosphohomoserine activation of the threonine
synthetase-associated threonine dehydratase, and demonstrates that threonine at this concentration in the presence of homoserine or phos0.015
-A
A 0~~~~
-0.010
=i0.005
10
o
150 200 250 300 350 SECONDS FIG. 5. Rate of reduction by lactate dehydrogenase of keto acid formed from homoserine. Keto acid was synthesized from 50 mM homoserine, 10 mM A TP, and 5 mM MgSO4 by incubation for 135 min in the presence of crude extract of ile-3, sprA-8, sprB-24. The amount of keto acid formed was determined colorimetrically. A sample of the 135-min reaction mixture containing 0.02 umol of keto acid (-) or 0.02 gmol of pyruvate (A) or a-ketobutyrate (0) in the zero-time reaction mixture was used as substrate in a reaction mixture containing 100 mMpotassium phosphate (pH 7.5), 0.1 mM NADH, and 0.1 unit of lactate dehydrogenase. 0
50
100
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SCHILDKRAUT AND GREER
782
TABLE 2. Radiochromatographic determination of the fate of 14C-D, L-homoserine and 'H- L-threoninea H counts/min
'4C counts/min
R,
Compound
Homoserine
0.43 0.05 0.58 0.33
.........................
Phosphohomoserine ................. a-Ketobutyrate ..................... Threonine ..........................
0 min
40 min
240 min
0 min
40 min
240 min
10,800 1,300
