Threonine synthetase-catalyzed conversion of phosphohomoserine to alpha-ketobutyrate in Bacillus...

JOURNAL OF BACrERIOLOGY, Sept. 1973, p. 777-785Copyright 0 1973 American Society for Microbiology

Vol. 115, No. 3Printed in U.S.A.

Threonine Synthetase-Catalyzed Conversion ofPhosphohomoserine to a-Ketobutyrate in

Bacillus subtilisIRA 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 thedephosphorylation and deamination of phosphohomoserine to a-ketobutyrate,resulting in a bypass of threonine in isoleucine biosynthesis. In crude extracts of astrain deficient in the biosynthetic isoleucine-inhibitable threonine dehydratase,phosphohomoserine was converted to a-ketobutyrate. Phosphohomoserine con-

version to a-ketobutyrate was shown not to involve a threonine intermediate.Single mutational events affecting threonine synthetase also affected the phos-phohomoserine-deaminating activity, suggesting that the deamination of phospho-homoserine was catalyzed by the threonine synthetase enzyme. It was demon-strated in vivo, in a strain deficient in the biosynthetic threonine dehydratase,that isoleucine was synthesized from homoserine without intermediate forma-tion of threonine.

Previous work in this laboratory (17) demon-strated that threonine synthetase (EC 2.4.99.2;phosphohomoserine _ threonine + inorganicphosphate) of Bacillus subtilis can also catalyzethe deamination of threonine to yield a-ketobu-tyrate. This result, and consideration of thecatalytic mechanisms of threonine synthetase ofNeurospora (7, 8) and the biodegradative threo-nine 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 anddeamination of phosphohomoserine to a-ketobutyrate without intermediate formation ofthreonine.The synthesis of threonine from phos-

phohomoserine proceeds by formation of an a,,unsaturated intermediate, a-aminocrotonate,to which water is added forming the pyridoxalSchiff base of threonine (7, 8). a-Aminocroton-ate is also an intermediate in the conversion ofthreonine to a-ketobutyrate catalyzed by threo-nine dehydratase (15). However, in the threo-nine dehydratase reaction, a-aminocrotonate iseliminated from the enzyme and is spontane-ously hydrolyzed to a-ketobutyrate and am-monia. It is proposed that if a-aminocrotonatewere eliminated from the threonine synthetaseenzyme, direct synthesis of a-ketobutyrate from

phosphohomoserine could occur without inter-mediate formation of threonine.

This paper presents evidence for an en-zymatic activity associated with threonine syn-thetase that catalyzes the dephosphorylationand deamination of phosphohomoserine. In ad-dition, evidence is provided for the in vivoconversion of homoserine to isoleucine withoutintermediate formation of threonine, the usu-ally recognized intermediate in isoleucine bio-synthesis. (A summary of this paper was pre-sented at the 73rd Annual Meeting of theAmerican Society for Microbiology, 6-11, May1973.)

MATERIALS AND METHODSBacterial-strains. Mutant strains of B. subtilis

(Table 1) were derived from strains 23 and 168 indole-of Burckholder and Giles (3) spontaneously or by invitro mutagenesis of deoxyribonucleic acid followedby transformation (20). All mutant strains utilized inthis study were deficient in the biosynthetic, isoleu-cine-inhibitable threonine dehydratase (17, 20, 21).The phenotypic expression of the ile mutation isisoleucine auxotrophy. The residual threonine dehy-dratase activity observed in these strains is known tobe an associated activity of threonine synthetase (17).Many of the strains used possessed either or both oftwo mutations that partially suppress the original ilemutation. One suppressor, sprA, results in a 5- to

777

SCHILDKRAUT AND GREER

TABLE 1. Isoleucine and threonine auxotrophicmutations and mutations of partial reversion to

prototrophy

Strain Amino acid requirement

ile-3 Isoleucineile-3, sprA-44 Isoleucine or homoserine or

threonineile-3, sprB-24 Isoleucine or homoserine or

threonineile-3, sprA-8, sprB-24 Noneile-3, sprA-44, thrB-37 Isoleucine plus threonineile-3, sprA-44, tdm-3 Isoleucineile-3, sprA-44, thrA-47 Threonine

10-f'old derepression of the threonine biosyntheticenzymes and therefore a 5- to 10-fold derepression of'the threonine synthetase-associated threonine dehy-dratase (17). The other suppressor, sprB, maps withinthe gene encoding threonine synthetase, and results ina 90% loss of' threonine synthetase activity, but noapparent change in the threonine synthetase-associated threonine dehydratase activity (17). Eithersuppressor mutation alone allows for growth of astrain on threonine or homoserine as well as isoleucine(20). Together, the two suppressor mutations result inprototrophy (20). The thrB mutation results in a lossof threonine synthetase and threonine synthetase-associated threonine dehydratase activity (17), tdmcauses a 90% decrease in threonine synthetase activityand a lack of threonine synthetase-associated threo-nine dehydratase activity (17), and thrA is a mutationthat results in a lack of' homoserine kinase activity(17).

Chemicals. Amino acids were obtained fromSigma Chemical Co. and Calbiochem. Alcohol dehy-drogenase, lactate dehydrogenase, deoxyribonuclease,and ribonuclease were obtained from Sigma ChemicalCo. Lysozyme (salt-free) was a product of Worthing-ton Biochemical Corp. Uniformly labeled '4C-L-threo-nine, '4C -L-methionine, and '4C-L-isoleucine, gener-ally labeled 3H-L-threonine, and 4- '4C-D, L-homose-rine were obtained from Schwarz/Mann. Uniformlylabeled '4C-L-homoserine was obtained from Amer-sham/Searle.

Synthesis of phosphohomoserine. Phos-phohomoserine was synthesized from homoserine byuse of' the homoserine kinase obtained from baker'syeast, by a modification of the method of' Flavin (6).Baker's yeast was obtained commercially and waswashed in 0.1 M tris(hydroxymethyl)aminomethane(Tris)-hydrochloride, pH 8.0. Disruption of yeast cellswas accomplished by treating 70 g (wet weight) in 90ml of' 0.1 M Tris-hydrochloride (pH 8.0) at 0 C in aSorvall Omnimixer at one-half' maximum speed for 20min with 210 g of' glass beads of' 100 to 200 umdiameter. The lysate was centrif'uged at 27,000 x g for15 min at 0 C, and the supernatant f'luid wasrecentrif'uged at 43,000 x g at 0 C for 30 min. Anammonium sulfate fraction was obtained between 30and 43% saturation. This fraction was washed in 45%saturated ammonium sulfate in 0.1 M Tris-hydrochlo-ride (pH 8.0) and was finally dissolved in 30 ml of 40

mM Tris-hydrochloride (pH 7.5) containing 4 mMreduced 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 triphos-phate (ATP), and 1 mmol of NaF in a total volume of'100 ml. The pH was adjusted to 7.5 with KOH, and 8ml of the yeast 30 to 43% ammonium sulfate fractionwas added to the reaction mixture. The reaction wasmonitored, and phosphohomoserine was purified ac-cording 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 followingcriteria to be phosphohomoserine. The compoundmigrated with an R, value described for phos-phohomoserine (17) in descending chromatographywith Whatman no. 52 paper and 88% phenol as asolvent. The compound gave coincident positive nin-hydrin and phosphate reactions. When the compoundwas chromatographed in 100-fold excess, no addi-tional ninhydrin spots were found, indicating that thecompound had less than 1% contaminating aminoacids. A quantitative ninhydrin (14) and quantitativebound phosphate determination (5, 10) gave a phos-phate to amino acid molar ratio of' 0.94. The phos-phate determination demonstrated that less than 1%inorganic phosphate was present. Incubation of thecompound in crude extracts of B. subtilis resulted inthe 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' theamino acid(s)/ml required by the particular strain.Cells were harvested in mid-log phase and washedonce with the buffer to be used in subsequent enzymeassays. The pellet was then resuspended in 10 to 20volumes of buffer, and the cell suspension was usedimmediately or frozen in portions of 1 to 2 ml at -30C. The specific activities of' the enzymes studied werethe same in fresh and frozen cells. Cells were lysedwith 200 Mg of lysozyme/mi at 37 C. Protein concen-tration 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 byUmbarger and Brown (19). The assay was carried outin a 0.5-ml volume containing 100 mM potassiumphosphate (pH 8.0), 0.1 mM pyridoxal phosphate,substrate (threonine, homoserine, or phosphohomoser-ine), and 0.2 to 1.0 mg of crude extract protein. Thereaction mixture was incubated at 37 C for theindicated times and was assayed for keto acidscolorimetrically by Greenberg's modif'ication (11) ofthe method described by Friedemann and Haugen(9). Monosodium a-ketobutyrate was the standard.Control tubes contained no substrate. The initial rateof a-ketobutyrate formation was proportional to pro-tein concentration.

Threonine synthetase. The method used was amodif'ication of' that described by Flavin (6). Theassay was carried out in a 1.25-mi volume containing0.6 mM phosphohomoserine, 0.08 mM pyridoxalphosphate, 80 mM potassium phosphate (pH 8.0),

778 J. BACTERIOL.

VOL. 115, 1973 PHOSPHOHOMOSERINE CONVERSION TO a-KETOBUTYRATE

and crude extract. Control tubes contained no phos-phohomoserine. The reaction was stopped by boilingfor 1 min, and the protein precipitate was removed bycentrifugation at 0 to 5 C. Threonine was assayed byperiodate oxidation to acetaldehyde, which was meas-ured by the amount of reduced nicotinamide adeninedinucleotide (NADH) oxidized in the presence ofalcohol dehydrogenase (6). Controls for the periodateoxidation consisted of homoserine, phosphohomose-rine, and a-ketobutyrate, each of which resulted inless than 1% oxidation of NADH as compared with thethreonine standard.Fate of "C-D, u.homoserine and 3H-ithreo-

nine. The radioactive metabolites present at 0, 40,and 240 min in a reaction mixture for enzymatic a-ketobutyrate formation containing 3H-L-threonine,"C-D, L-homoserine, and ATP were analyzed bydescending chromatography. Samples of the reactionmixture (10 ,liters) were applied to Whatman no. 52(acid-washed) filter paper, and 88% phenol was uti-lized as the solvent. Amino acid and a-ketobutyratestandards were chromatographed in parallel with thesamples on each chromatogram, and the amino acidstandards were identified by ninhydrin reagent spray.Phosphohomoserine, in addition to a positive ninhy-drin test, gave a positive reaction in the phosphatetest. a-Ketobutyrate was demonstrated with bromo-cresol green spray (2). As an additional control,'4C-D, L-homoserine and 3H-L-threonine were chroma-tographed in the presence of unlabeled reaction mix-ture. The chromatograms were dried at room temper-ature and cut into fractions. Each fraction was elutedwith water and counted in a liquid scintillationspectrometer with correction made for spillover be-tween 14C and 3H channels.

Conversion of homoserine to isoleucine in vivo.Strain ile-3, sprB-24 was grown in unlabeled mediumto mid-log phase and then inoculated into 5 ml ofidentical medium containing "4C-labeled amino acid.At the end of log growth, which corresponded toapproximately two generations in labeled medium,the culture was centrifuged and washed in 0.1 Mpotassium phosphate buffer (pH 8.0), lysed with 200ug of lysozyme/ml, and treated with 500 ,g of ribonu-clease/ml and 30 gg of deoxyribonuclease/ml. Theprotein was precipitated and washed three times with100% saturated ammonium sulfate at 0 C. The pelletwas dissolved in 5.0 ml of water. A 2-ml sample wasdialyzed overnight against two changes of 3 liters ofwater. The protein was hydrolyzed in 6 N HCl byheating to 110 C for 12 h. The hydrolysate was driedand redissolved in 0.1 ml of water; 10 Mliters wasapplied to Whatman 3MM paper, dried, and chroma-tographed with butanol-acetic acid-water, 12:5:3.The paper was dried, and the distribution of radioac-tivity was determined with a Baird Atomic radio-chromatogram scanner. "4C-labeled standards wererun through the hydrolysis procedure and used ascontrols in the chromatographic system. The relativeamounts of radioactivity were determined by inte-grating the areas under the peaks of the radioactivityscan. Counts per minute per radioactive area wereobtained by cutting out radioactive areas and count-ing by liquid scintillation spectroscopy.

RESULTSConversion of homoserine and phosphoho-

moserine to a-ketobutyrate. In crude extractsof strains lacking the biosynthetic isoleucineend product-inhibitable threonine dehydratase,a-ketobutyrate formation from homoserine or

phosphohomoserine occurred at a rate similar tothat obtained with threonine (Fig. 1). Theformation of a-ketobutyrate from phos-phohomoserine was linear with time and proteinconcentration (Fig. 2). Since all strains utilizedin this study lacked the biosynthetic isoleucine-inhibitable threonine dehydratase, the only re-

maining threonine dehydratase activity was thethreonine synthetase-associated threoninedehydratase (17). In the case of homoserine,ATP was added to the reaction mixture so thathomoserine could be converted to phosphoho-moserine by homoserine kinase (EC 2.7.1.39).The conversion of homoserine to a-ketobutyratedid not occur when ATP was not added to thereaction mixture, although a-ketobutyrate was

formed from phosphohomoserine (Fig. 3). Fur-thermore, a mutant strain lacking homoserine

0.4-

~~~~50 NOMISERINE (+ATPJ

~0.3-c-

__ m*N TNREONINE

0.2m

Lhi

.5~~~~~v mM PNOSPNONSMOSERINE

MINUTESFIG. 1. Conversion of homoserine, phos-

phohomoserine, and threonine to a-ketobutyrate.Homoserine, 50 mM, plus 10 mM ATP and 5 mMMgSO4 (A), phosphohomoserine, 5 mM (0), orthreonine, 50 mM (0), was incubated in the presenceof crude extract of ile-3, sprA-8, sprB-24. The forma-tion of a-ketobutyrate was determined colorimetri-cally.

779

standard on Whatman no. 52 chromatographypaper with 88% phenol as solvent (Table 2).

0.8- Conversion of phosphohomoserine to a-ke-tobutyrate without intermediate threonineformation. The conversion of phosphohomoser-

W / ine to a-ketobutyrate could occur via a two-step catalytic process, threonine synthetase and

-0.6/ the threonine synthetase-associated threoninedehydratase, or by the alternatively proposed

/ direct dephosphorylation and deamination of-/* phosphohomoserine. To eliminate the possibil-

ity that phosphohomoserine is converted to*./ 2 a-ketobutyrate via a threonine intermediate, we

Y 0.4- ~ / / assayed the formation of a-ketobutyrate fromlow concentrations of phosphohomoserine andthreonine in crude extract. The intention of

0*/° lowering the substrate concentration was to/o/D demonstrate a difference in the Km for a-

- 0.2 - ketobutyrate formation from phosphohomoser-/ mine and threonine. Since the Km for threonine

of the threonine synthetase-associated threo-o//: , nine dehydratase activity is large, approxi-

mately 50 mM (17), a low concentration of phos-phohomoserine might be converted to a-ketobu-

0 60 120 180 tyrate at a faster rate than a low concentrationMINUTES of threonine.

FIG. 2. Formation of a-ketobutyrate as a function The rate of a-ketobutyrate formation from 2.5of time and protein concentration. Phosphohomoser- mM phosphohomoserine was at least 20-foldine, 5 mM, was incubated in the presence of crudeextract of ile-3, sprA-8, sprB-24. The formation of 0.15-a-ketobutyrate was determined colorimetrically. Re-action vessels contained 1.6 mg (0), 0.80 mg (0), or0.40 mg (0) of protein per ml of reaction volume.

2.5 mM PNISPNONOSMSEUINEkinase was unable to convert homoserine to /a-ketobutyrate, indicating that phos- ,phohomoserine is a necessary intermediate for 0.10lthe formation of a-ketobutyrate from homo- - /serine (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 phos-phohomoserine was a-ketobutyrate. A sample 0.05of a reaction mixture in which keto acid was °produced from homoserine in the presence of M

crude extract, and ATP was incubated withlactate dehydrogenase and NADH. The rate ofoxidation of NADH to NAD with the keto acidformed enzymatically from homoserine was Es ImIUNSEIINE V-ATPJsimilar to that observed with a-ketobutyrate, Isio16 23 30 46 5I 6I 7ii 0 9rather than with pyruvate (Fig. 5). This indi- MINUTEScates that the keto acid formed is not pyruvate FIG. 3. Lack of conversion of homoserine to a-and is consistent with the formation of a- ketobutyrate when ATP is excluded from the reactionketobutyrate. Furthermore, 14C-D, L-homoser- mixture. Homoserine, 5 mM (0), or phosphohomoser-ine incubated with crude extract and ATP was ine, 2.5 mM (0), was incubated in the presence ofconverted to a "4C-labeled product that mi- crude extract of ile-3, sprA-8, sprB-24. The formationgrated coincident with an a-ketobutyrate of a-ketobutyrate was determined colorimetrically.

SCHILDKRAUT AND GREER J. BACTERIOL.780

VOL. 115, 1973 PHOSPHOHOMOSERINE CONVERSION TO a-KETOBUTYRATE

0.2

5 mu PHOSPIOHIMOSCRINE/~~~~~~~0.1 /

5 mN NONOSERINE (+ATPJO 0Q 0

15 30 45 60MINUTES

FIG. 4. Lack of conversion of homoserine to a-

ketobutyrate in a strain lacking homoserine kinase.Homoserine, 5 mM, plus 10 mM ATP and 5 mMMgS04 (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-ketobutyratewas determined colorimetrically.

higher than the rate from 2.5 mM threonine(Fig. 6). The rate of conversion of 25 mMphosphohomoserine was twofold higher than therate from 25 mM threonine. At a low concentra-tion of threonine, the rate of a-ketobutyrateformation was substantially lower than at a

high threonine concentration (Fig. 6), as was

expected because of the high Km of the threo-nine synthetase-associated threonine dehydra-tase for threonine. Since a low concentration ofphosphohomoserine was converted to a-ketobu-tyrate more readily than was threonine, thesynthesis of a-ketobutyrate from phos-phohomoserine may occur without intermediatethreonine formation. This is consistent with thesuggestion that there is direct conversion ofphosphohomoserine to a-ketobutyrate. It is con-ceivable, however, that phosphohomoserine ac-

tivates the threonine synthetase-associatedthreonine dehydratase activity by lowering theKm for threonine. If this were the case, lowconcentrations of threonine could be efficientlyconverted to a-ketobutyrate in the presence ofphosphohomoserine.To rule out this possibility, we performed an

experiment in which both homoserine andthreonine were present in the same incubation

mixture. The fate of "4C-D,L-homoserine and3H-L-threonine was determined by chromatog-raphy of samples of the reaction mixture aftervarious times of incubation (Table 2). Theconcentrations of substrates were chosen so thatconversion of threonine to a-ketobutyrate viathe threonine synthetase-associated threoninedehydratase activity would occur at a reducedrate, unless the threonine synthetase-associatedthreonine dehydratase activity was activated byphosphohomoserine. There was a rapid conver-sion of "C-homoserine to 04C-phosphohomoser-ine. Some "4C-phosphohomoserine is found inthe zero-time reaction mixture, probably as aresult of the time it takes to apply the reactionmixture and dry it on the paper. At later times,the 04C-phosphohomoserine is converted to 14C_threonine and "4C-a-ketobutyrate in an approx-imate molar ratio of 2: 1. It should be noted thatapproximately one-half of the starting homo-serine remains unaltered during the incubation;this corresponds to the D isomer of homoserine.In contrast to the conversion of phosphohomo-serine to a-ketobutyrate, the rate of formationof a-ketobutyrate from 3H-threonine was lessthan 3% of the rate from phosphohomoserine.This experiment rules out the possibility of

phosphohomoserine activation of the threoninesynthetase-associated threonine dehydratase,and demonstrates that threonine at this concen-tration in the presence of homoserine or phos-

0.015 A -A

0~~~~

-0.01010

=i0.005 o

0 50 100 150 200 250 300 350SECONDS

FIG. 5. Rate of reduction by lactate dehydrogenaseof keto acid formed from homoserine. Keto acid wassynthesized from 50 mM homoserine, 10 mM A TP,and 5 mM MgSO4 by incubation for 135 min in thepresence of crude extract of ile-3, sprA-8, sprB-24.The amount of keto acid formed was determinedcolorimetrically. A sample of the 135-min reactionmixture containing 0.02 umol of keto acid (-) or 0.02gmol of pyruvate (A) or a-ketobutyrate (0) in thezero-time reaction mixture was used as substrate in areaction mixture containing 100 mMpotassium phos-phate (pH 7.5), 0.1 mM NADH, and 0.1 unit oflactate dehydrogenase.

781

SCHILDKRAUT AND GREER

TABLE 2. Radiochromatographic determination of the fate of 14C-D, L-homoserine and 'H- L-threoninea

'4C counts/min H counts/minCompound R,

0 min 40 min 240 min 0 min 40 min 240 min

Homoserine ......................... 0.43 10,800 5,500 5,250 <50 <50 <50Phosphohomoserine ................. 0.05 1,300 5,400 1,500 <50 <50 <50a-Ketobutyrate ..................... 0.58 <50 100 1,400 < 50 <50 < 50Threonine .......................... 0.33 <50 300 2,850 18,200 18,000 17,000

a 14C-D,L-homoserine (5.0 mM, 0.2 gCi/Umol), 3H-L-threonine (2.5 mM, 2 gCi/gmol), ATP (5 mM), andMgSO4 (2.5 mM) were incubated together in the presence of crude extract of ile-3, sprA-8, sprB-24. Thereaction mixture was chromatographed at 0, 40, and 240 min.

25 mm

a= 0.4 ; PHOSPWONOMOSERINE_ 0.4 S

0~~~~

/25 1TNIEGNINE0.2-

~ ~ ~ ~ ~ ~

30 60 90MINUTES

FIG. 6. Conversion of phosphohomoserine andthreonine to a-ketobutyrate. Phosphohomoserine, 25mM (0) or 2.5 mM (0), and threonine, 25 mM (-) or

2.5 mM (0) were incubated in the presence of crudeextract of ile-3, sprA-8, sprB-24. The formation ofa-ketobutyrate was determined colorimetrically.

phohomoserine is not converted at an apprecia-ble rate to a-ketobutyrate; therefore, a-ketobu-tyrate is derived directly from phosphohomoser-ine. These results indicate that there exists incrude extracts of B. subtilis an enzyme whichconverts phosphohomoserine to a-ketobutyrate.This enzyme activity is referred to here as

phosphohomoserine deaminase.Association of phosphohomoserine deam-

inase with threonine synthetase. To demon-strate that the phosphohomoserine deaminaseactivity is associated with threonine synthetase,we determined the specific enzymatic activitiesof various mutant strains of B. subtilis. As can

be seen from the data presented in Table 3, a

single mutational event (thrB-37 or tdm-3)

which affects threonine synthetase or its as-sociated threonine dehydratase activity alsoaffects the phosphohomoserine deaminase ac-tivity. Furthermore, phosphohomoserine deam-inase is coordinately derepressed (sprA) withthreonine synthetase and its associated threo-nine dehydratase activity. These data suggestthat phosphohomoserine deaminase is a cata-lytic activity of threonine synthetase, as is thethreonine synthetase-associated threoninedehydratase.

In vivo evidence for phosphohomoserinedeaminase activity. Evidence that threoninecould be bypassed in the biosynthesis of isoleu-cine from homoserine would lend support to anin vivo role for phosphohomoserine deaminase.Strain ile-3, sprB-24 was grown in the presenceof 3 ,ug of 4C-L-homoserine/ml (3 jCi/4mol) andexcess unlabeled threonine (100 ,ug/ml). If iso-leucine is derived exclusively from threonine,then radioactive label derived from homoserineshould be diluted as a result of the presence ofunlabeled threonine to the same extent inisoleucine and threonine residues in cellularprotein. However, if isoleucine is not derivedexclusively from the intracellular threoninepool, but in addition is derived from homoserinethat bypassed a threonine intermediate, thedilution of radioactivity in isoleucine shouldoccur to a lesser extent than dilution of radioac-tivity in threonine. The '4C-isoleucine to 14C-threonine ratio found in protein was 3.8, incontrast to a ratio of 1.3 of isoleucine to threo-nine determined from B. subtilis protein (18).The ratio of 1.3 would be predicted if thepathway for the conversion of homoserine toisoleucine was via a threonine intermediate.The result of this experiment indicates thathomoserine is converted to isoleucine withoutintermediate formation of threonine.

In another experiment, the same strain of B.subtilis was grown in the presence of '4C-threo-nine and increasing concentrations of unlabeledhomoserine (Table 4). If the only pathway forconversion of homoserine to isoleucine is via athreonine intermediate, then the radioactive

782 J. BACTERIOL.

PHOSPHOHOMOSERINE CONVERSION TO a-KETOBUTYRATE

TABLE 3. Effect of mutations on threonine synthetase, its associated threonine dehydratase, andphosphohomoserine deaminase activity

Specific activity (gmol per h per mg of protein)

Strain Phosphohomoserine deaminasecThreonine Threoninesynthetasea dehydrataseb Homoserine Phos-

+ ATP phohomoserine

ile-3.0.82 0.04 0.03 0.02ile-3,sprA-44 ........................... 6.0 0.41 0.34 0.25ile-3, sprB-24 ........................... 0.08 0.05 0.06 0.06ile-3, sprA-8, sprB-24 .......... ......... 0.54 0.30 0.20 0.30ile-3, sprA-44, thrB-37 ......... ......... <0.01 0.03 0.01 0.02ile-3, sprA-44, thrA-47 ......... ......... 4.7 0.33 0.Old 0.36ile-3, sprA-44, tdm-3 .................... 0.49 <0.01 0.01 <0.01

a Determined by Skarstedt and Greer (17).b Threonine dehydratase activity is a measure of the threonine synthetase-associated threonine dehydratase

activity, since the biosynthetic isoleucine end product-inhibitable threonine dehydratase is absent in all casesas a result of the ile mutation. The determination of specific activity was made with 50 mM threonine assubstrate.

c Phosphohomoserine deaminase was assayed with 50 mM homoserine, 10 mM ATP, and 5 mM MgSO4, orwith 5 mM phosphohomoserine as substrate.

d This strain contains a mutation in the gene encoding homoserine kinase, thrA-47; therefore, one does notexpect to observe phosphohomoserine deaminase activity with homoserine and ATP.

threonine incorporated into protein threonineand isoleucine should be diluted to equal ex-

tents in the presence of excess unlabeled homo-serine. However, if homoserine is converted toisoleucine without intermediate formation ofthreonine, the presence of unlabeled homo-serine should result in greater dilution of theradioactivity incorporated into protein isoleu-cine than into threonine. When 100 ug of"4C-threonine/ml was present and no homo-serine was added, the ratio of "4C-isoleucine to"4C-threonine in protein was approximately 1.0,which is similar to the total protein isoleucine tothreonine ratio of 1.3 (18). This small discrep-ancy may be due to direct conversion of endoge-nous homoserine to isoleucine without interme-diate formation of threonine. At the homoserineconcentration of 3 Asg/ml, there was no signifi-cant change in the ratio of "4C-isoleucine to"4C-threonine. However, at equal concentra-tions of unlabeled homoserine and "4C-threo-nine (100 1sg/ml), the ratio declined significantlyto 0.26. Dilution of "IC-labeled isoleucine byunlabeled homoserine was greater than dilutionof "4C-labeled threonine. These experimentsestablish that isoleucine may be synthesizedfrom homoserine in vivo without a threonineintermediate.

DISCUSSIONThe above results demonstrate that B. sub-

tilis has an enzymatic activity, termed phos-phohomoserine deaminase, which catalyzes thedephosphorylation and deamination of phos-

TABLE 4. Effect of unlabeled homoserine on14C-threonine incorporation into protein threonine

and isoleucine residuesa

Concn of un-14C counts/min Ratio of '4C-iso-labeled L-homo- leucine to

serine in growth Isoleucine Threonine 14C-threoninemedium (gg/mi)0 2,460 2,650 0.933 2,525 2,750 0.92

100 700 2,650 0.26

aProtein hydrolysates of ile-3, sprB-24 were madeand chromatographed after the strain was grown inmedium containing 100 jAg of "4C-L-threonine per ml(1 ACi/Ijmol) and various concentrations of unlabeledhomoserine.

phohomoserine to yield a-ketobutyrate. To ourknowledge, this enzymatic activity has notpreviously been observed or postulated. Geneticevidence suggests an association of this activitywith the threonine synthetase protein.The reaction catalyzed by phosphohomoser-

ine deaminase is equivalent to the sum of thetwo reactions catalyzed by the two enzymesthreonine synthetase and threonine dehydra-tase. Synthesis of a-ketobutyrate by phos-phohomoserine deaminase, however, does notrequire intermediate threonine formation, asdoes the two-step conversion of phos-phohomoserine to a-ketobutyrate catalyzed bythreonine synthetase and threonine dehydra-tase.Independent investigations have shown that

783VOL. 115, 1973

SCHILDKRAUT AND GREER

threonine synthetase and threonine dehydra-tase form an identical enzyme-bound interme-diate, a-aminocrotonate (7, 8, 15). The forma-tion of a-ketobutyrate by the threonine synthe-tase enzyme of B. subtilis may occur via spon-taneous hydrolysis of a-aminocrotonate re-leased from the enzyme, as occurs in the forma-tion of a-ketobutyrate catalyzed by threoninedehydratase. a-Aminocrotonate is known also(12, 16) to be a reaction intermediate in thereactions catalyzed by cystathionine synthase(EC 4.2.1.21). Succinyl-homoserine reactswith cystathionine synthase to form the pyrid-oxal-bound intermediate a-aminocrotonate. Inthe presence of cysteine, this intermediate willreact to form cystathionine and succinate.However, in the abence of cysteine, the a-aminocrotonate intermediate is eliminated fromthe enzyme and spontaneously hydrolyzes toform a-ketobutyrate. It is of interest to notethat DeLavier-Klutchko and Flavin (4) sug-gested that the formation of a-ketobutyratecould not be observed with threonine synthetasebecause the attacking group for threonine for-mation is water and cannot be excluded from thereaction mixture, as could be done with cysteinein the case of cystathionine synthase. However,it is proposed here that the final product formedfrom phosphohomoserine by threonine synthe-tase of B. subtilis will be either threonine ora-ketobutyrate, depending upon whether thea-aminocrotonate intermediate is eliminatedfrom the enzyme and spontaneously hydrolyzedto a-ketobutyrate or whether the intermediateremains bound and is subjected to nucleophilicattack by water, at the d-carbon atom, to formthreonine.

Previous work in this laboratory (17, 20, 21)led to the suggestion that derepression of thethreonine synthetase-associated threoninedehydratase activity could be a mechanism ofsuppression of isoleucine auxotrophs deficientin the biosynthetic threonine dehydratase.However, the present results suggest that sup-pression of isoleucine auxotrophy may be inter-preted in terms of an increase in phos-phohomoserine deaminase activity. An impor-tant distinction can be made between conver-sion of phosphohomoserine to a-ketobutyratevia a threonine intermediate (threonine synthe-tase and its associated threonine dehydrataseactivity) and the direct conversion of phos-phohomoserine to a-ketobutyrate (phosphoho-moserine deaminase). Since low concentrationsof phosphohomoserine are converted to a-keto-butyrate more readily than low concentrationsof threonine, the affinity of this enzyme forthreonine is apparently lower than its affinityfor phosphohomoserine. Phosphohomoserine

deaminase activity does not involve the forma-tion of threonine and, therefore, does not re-quire the deamination of threonine for whichthe threonine synthetase-associated threoninedehydratase has a low affinity.Homoserine was shown to bypass threonine in

the biosynthesis of isoleucine for protein, indi-cating that phosphohomoserine deaminase didcatalyze the direct conversion of phos-phohomoserine to a-ketobutyrate in vivo. Thesestudies were performed with a mutant strainlacking the biosynthetic threonine dehydrataseand containing the sprB mutation. An in vitrodetermination in this strain of the ratio of thetwo activities, threonine synthetase and phos-phohomoserine deaminase, demonstrated thatthreonine and a-ketobutyrate were formed in amolar ratio of 2: 1. However, with the wild-typethreonine synthetase, the ratio of the activitiesis approximately 20: 1 under the in vitro assayconditions employed. Since the wild-type strainalso has the biosynthetic isoleucine end prod-uct-inhibitable threonine dehydratase, theevidence presented does not necessarily imply arole for phosphohomoserine deaminase in thewild-type strain. Work is in progress to investi-gate further the biological significance of phos-phohomoserine deaminase.

Threonine synthetase of B. subtilis may be auseful model system for studying the mech-anisms of pyridoxal catalysis. Alteration ofthreonine synthetase by mutation can changeits catalytic properties and affect the ratio ofphosphohomoserine deaminase to threoninesynthetase activity. For example the sprB mu-tation decreases threonine synthetase activity90%, whereas phosphohomoserine deaminaseactivity is relatively unaltered. The use of suchmutants may contribute to the elucidation ofthe mechanism responsible for the conversion ofphosphohomoserine to two different products,threonine or a-ketobutyrate.

ACKNOWLEDGMENT

We thank Geoffrey Cooper for many helptul discussions,advice, and critical reading of the manuscript.

LITERATURE CITED

1. Anagnostapoulos, C., and J. Spizizen. 1961. Require-ments for transformation in Bacillus subtilis. J. Bac-teriol. 81:741-746.

2. Bobbitt, J. M. 1961. Thin-layer chromatography, p. 88.Reinhold Publishing Corp., New York.

3. Burckholder, P. R., and N. H. Giles. 1947. Inducedbiochemical mutations in Bacillus subtilis. Amer. J.Bot. 34:345-348.

4. DeLavier-Klutchko, C., and M. Flavin. 1965. Enzymaticsynthesis and cleavage of cystathionine in fungi andbacteria. J. Biol. Chem. 240:2537-2549.

5. Fiske, C. H. and Y. SubbaRow. 1925. The colorimetricdetermination of phosphorus. J. Biol. Chem.66:375-400.

784 J. BACTERIOL.

VOL. 115, 1973 PHOSPHOHOMOSERINE CONVERSION TO a-KETOBUTYRATE

6. Flavin, M. 1961. Threonine synthetase from Neurospora,p. 951-959. In S. P. Colowick and N. 0. Kaplan (ed.),Methods in enzymology, vol. 5. Academic Press Inc.,New York.

7. Flavin, M., and T. Kono. 1960. Threonine synthetasemechanism: studies with isotopic oxygen. J. Biol.Chem. 235:1109-1111.

8. Flavin, M., and C. Slaughter. 1960. Threonine synthetasemechanism: studies with isotopic hydrogen. J. Biol.Chem. 235:1112-1118.

9. Friedemann, T. E., and G. E. Haugen. 1943. Pyruvicacid. II. The determination of keto acids in blood andurine. J. Biol. Chem. 147:415-442.

10. Gomori, G. 1942. A modification of the colorimetricphosphorus determination for use with the photoelec-tric colorimeter. J. Lab. Clin. Med. 27:955-960.

11. Greenberg, D. M. 1962. Cystathionine and homoserinecleavage, p. 936-947. In S. P. Colowick and N. 0.

Kaplan (ed.), Methods in enzymology, vol. 5. Aca-demic Press Inc., New York.

12. Kaplan, M., and M. Flavin. 1966. Cystathionine -y-syn-thase of Salmonella, catalytic properties of a newenzyme in bacterial methionine biosynthesis. J. Biol.Chem. 241:4463-4471.

13. Lowry, 0 H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the folinphenol reagent. J. Biol. Chem. 193:265-275.

14. Moore, S., and W. H. Stein. 1954. A modified ninhydrin

reagent for the photometric determination of aminoacids and related compounds. J. Biol. Chem.211:907-913.

15. Phillips, A. T., and W. Wood. 1965. The mechanism ofaction of 5'-adenylic acid-activated threonine dehydra-tase. J. Biol. Chem. 240:4703-4709.

16. Posner, B., and M. Flavin. 1972. Cystathionine y-syn-thase, studies of hydrogen exchange reactions. J. Biol.Chem. 247:6402-6411.

17. Skarstedt, M., and S. Greer. 1973. Threonine synthetaseof Bacillus subtilis: the nature of an associated dehy-dratase activity. J. Biol. Chem. 248:1032-1044.

18. Stokes, J., and M. Gunness. 1946. The amino acidcomposition of microorganisms. J. Bacteriol.52:195-207.

19. Umbarger, H. E., and B. Brown. 1958. Isoleucine andvaline metabolism in Escherichia coli. VII. A negativefeedback mechanism controlling isoleucine biosynthe-sis. J. Biol. Chem. 233:415-420.

20. Vapnek, D., and S. Greer. 1971. Suppression by derepres-sion in threonine dehydratase-deficient mutants ofBacillus subtilis. J. Bacteriol. 106:615-625.

21. Vapnek, D., and S. Greer. 1971. Minor threonine dehy-dratase encoded within the threonine synthetic regionof Bacillus subtilis. J. Bacteriol. 106:983-993.

22. Wormser, E., and A. Pardee. 1958. Regulation of threo-nine biosynthesis in Escherichia coli. Arch. Biochem.Biophys. 78:416-432.

785