role of acetohydroxy acid isomeroreductase biosynthesis acid · 260 primerano andburns lactate,...

JOURNAL OF BACTERIOLOGY, Jan. 1983, p. 259-269 Vol. 153, No. 10021-9193/83/010259-11$02.00/0Copyright 0 1983, American Society for Microbiology

Role of Acetohydroxy Acid Isomeroreductase in Biosynthesisof Pantothenic Acid in Salmonella typhimurium

DONALD A. PRIMERANO AND R. 0. BURNS*Department ofMicrobiology and Immunology, Duke University School of Medicine, Durham, North Carolina

27710

Received 2 August 1982/Accepted 11 October 1982

Structural genes have been identified for all of the enzymes involved in thebiosynthesis of pantothenic acid in Salmonella typhimurium and Escherichia coliK-12, with the exception of ketopantoic acid reductase, which catalyzes theconversion of a-ketopantoate to pantoate. The acetohydroxy acid isomeroreduc-tase from S. typhimurium efficiently bound a-ketopantoate (Km = 0.25 mM) andcatalyzed its reduction at 1/20 the rate at which ot-acetolactate was reduced. Sincetwo enzymes could apparently participate in the synthesis of pantoate, a S.typhimurium ilvC8 strain was mutagenized to derive strains completely blocked inthe conversion of a-ketopantoate to pantoate. Several isolates were obtained thatgrew in isoleucine-valine medium supplemented with either pantoate or pantothe-nate, but not in the same medium supplemented with a-ketopantoate or P-alanine.The mutations that conferred pantoate auxotrophy (designated panE) to theseisolates appeared to be clustered, but were not linked to panB or panC. All panEstrains tested had greatly reduced levels of ketopantoic acid reductase (3 to 12% ofthe activity present in DU201). The capacity of the isomeroreductase to synthe-size pantoate in vivo was assessed by determining the growth requirements ofilvC+ derivatives ofpanE ilvC8 strains. These strains required either a-ketopan-toate, pantoate, or pantothenate when the isomeroreductase was present at lowlevels; when the synthesis of isomeroreductase was induced, panE ilvC+ strainsgrew in unsupplemented medium. These phenotypes indicate that a high level ofisomeroreductase is sufficient for the synthesis of pantoate. panE ilvC+ strainsalso grew in medium supplemented with lysine and methionine. This phenotyperesembles that of some S. typhimurium ilvG mutants (e.g., DU501) which arepartially blocked in the biosynthesis of coenzyme A and are limited for succinylcoenzyme A. panE ilvC+ strains which lack the acetohydroxy acid synthasesrequired only methionine for growth (in the presence of leucine, isoleucine, andvaline). This and other evidence suggested that the synthesis of pantoic acid byisomeroreductase was blocked by the aL-acetohydroxy acids and that pantoic acidsynthesis was enhanced in the absence of these intermediates, even when theisomeroreductase was at low levels. panE ilvC+ strains reverted to pantothenateindependence. Several of these revertants were shown to have elevated isomeror-eductase levels under noninduced and induced conditions; the suppressingmutation in each revertant was shown to be closely linked to ilvC by P22transduction. This procedure presents a means for obtaining mutants with alteredregulation of isomeroreductase.

Pantoic acid, an intermediate in the panto- The absence of mutants deficient in this reactionthenic acid biosynthetic pathway, is formed by suggests that more than one enzyme is able tothe reduction of a-ketopantoic acid (5, 13) (Fig. catalyze the conversion of oa-ketopantoate to1). This reaction is apparently catalyzed by pantoate.ketopantoic acid reductase in Escherichia coli A candidate for such an enzyme is acetohy-K-12 (12, 25, 26). Although structural genes for droxy acid isomeroreductase, the product of theall of the other pantothenic acid biosynthetic ilvC gene. This enzyme catalyzes the secondenzymes have been identified and mapped in E. common step in isoleucine and valine biosynthe-coli K-12 and in Salmonella typhimurium, the sis, i.e., the formation of a,3-dihydroxy-,B-gene encoding ketopantoic acid reductase has methylvalerate and Oa,,-dihydroxyisovaleratenot been identified in either species (2, 7, 8, 10). from a-aceto-oa-hydroxybutyrate and a-aceto-

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260 PRIMERANO AND BURNS

lactate, respectively (Fig. 1 and 2). The conver-sion of the a-acetohydroxy acids to these in-termediates is initiated with the migration of theaL-alkyl group of the acetohydroxy acid to the 1-carbon to form the a-keto-,-hydroxy acid whichis subsequently reduced to the a,R-dihydroxyacid by using the cosubstrate NADPH (Fig. 2).The isomerized intermediates, a-keto-,B-hy-droxy-13-methylvalerate and a-keto-,B-hy-droxyisovalerate, bear striking resemblance toa-ketopantoate (a-keto-P,B-dimethyl--y-hydrox-ybutyrate, Fig. 2). In the work presented herewe show that purified isomeroreductase cata-lyzes the reduction of a-ketopantoate and thatmutation in a gene which we designate panEleads to a requirement for pantoate when pre-sent in strains bearing an ilvC mutation. Condi-tions are described under which panE ilvC+strains grow in medium lacking pantothenate orits specific precursors.

MATERIALS AND METHODSChemicals and enzyme assays. Sodium a-ketoisoval-

erate, DL-pantoyl lactone, D-pantoyl lactone, and cal-cium D-pantothenate were obtained from the SigmaChemical Co., St. Louis, Mo. a-Ketopantoyl lactonewas prepared from DL-pantoyl lactone by the methodof Lipton and Strong (15). Potassium ax-ketopantoateto be used in either enzyme or nutritional studies wasprepared by titrating solutions of oa-ketopantoyl lac-tone to pH 7.5 with KOH. Potassium D-pantoate wasprepared by mixing 100mM D-pantoyl lactone with anequimolar amount of KOH and allowing the solutionto stand for 5 h to complete the hydrolysis. Sodium a-aceto-oL-hydroxybutyrate and sodium a-acetolactatewere prepared by saponification of the methyl esters,which were the generous gifts of Frank Armstrong.Acetohydroxy acid isomeroreductase was purified

threonine

| ThreonineNH31 deaminase WM)

a-k tobutyrote_, 0- a-aceto-a-hvdrox

from S. typhimurium by the method of Hofier et al.(11) and was assayed by the method of Arfin andUmbarger (1). Isomeroreductase assays used for thedetermination of kinetic constants were performedwith purified enzyme at 30°C; all other isomeroreduc-tase assays were performed at room temperature. a-Aceto-a-hydroxybutyrate was used exclusively as thesubstrate in the determination of isomeroreductaselevels in crude extracts. When the isomeroreductase incrude extract was tested for the ability to catalyze D-pantoate synthesis, 5 mM potassium a-ketopantoate(pH 7.5) was added to the standard reaction mixture inplace of a-aceto-a-hydroxybutyrate. One unit of iso-meroreductase activity is defined as the amount ofenzyme required to oxidize 1 nmol ofNADPH per minin the standard assay.Threonine deaminase (6), ketopantoate hydroxy-

methyltransferase (20, 24), and acetohydroxy acidsynthases I and II (23) were assayed as previouslydescribed; acetohydroxy acid synthase II was mea-sured in reaction mixtures containing 1 mM valine.Ketopantoic acid reductase was assayed by the meth-od of King and Wilken (13), except that the pH of thesodium acetate buffer was raised from 5.0 to 5.5. Theactivity of ketopantoyl lactone reductase in crudeextracts was estimated by the method of Wilken et al.(26).

Cell-free extracts were prepared from batch culturesas previously reported (3, 20). Protein concentrationswere determined by the method of Lowry et al. (16).

Characterization of product of isomeroreductase re-action with a-ketopantoate as substrate. Acetohydroxyacid isomeroreductase was observed to catalyze theoxidation of NADPH in the presence of a-ketopantoicacid. The (reduced) product of this reaction wascharacterized (i) by feeding the product to a series ofmutants blocked in consecutive steps of the panto-thenic acid pathway and (ii) by gas-liquid chromatog-raphy of the lactonized substrate and product alongwith authentic ketopantoyl lactone and D-pantoyl lac-tone.

.-kkoglutarat

ollomtisoloucine

keto-,B-mthvl rrnsomninase E ( ilvE I

NADPH NADP 420

Y- t /> ,8-dihydroxv-8-' 2bucoraze methylvalerate valerate

A (Ailvi) Acefohydroxy acid .klguaaoVlnPyruvate AHAS s(i/v@) Aocerorductasie Dihydroxy acid -keogiutorot,Valisei°C°2 A VAS3r UW) (idvC) dehydrase (i/D) glut oruv ate

Pyruvate a -acetolactate a,a ddihydroxy- - -. a- hketoisovalerate ann Traneamirose C (ovrA)i3OVisovlrato

NADPH NADP H20 AcetylKetopontoote CH2THF CoA a- osopropylmalotehydroxymethyltratensfrase Synthose U/euA)

THf CoAketopantoate a- isopropylmolate

Ketopontoic acid reductase (panE) NADPHor a-I opropylMOlato

Acetohydroxy acid isomororeductase (i/vC) NADP ilsomeraso (leuC, D)pantoate -isopropyimatate

aspartate-*B -aaanine ATP NADHNAspartatewl- Pantothenate _ 8-eopropymaelatD

decarboxylase synthelase(ponC) Ppi C02. NAD dehydroenasc (/uD)(panD) pontothenate a-ketois caproat

alutamata Transaminose B (f/rE)J or

.-ketoslutorat, Tyrocine aminotransterasecoenzyme A Leucine (yrD)

acyl Carrier protein

FIG. 1. Biosynthetic pathways of the branched-chain amino acids and pantothenic acid.

-a-I

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CATALYSIS OF PANTOIC ACID SYNTHESIS 261

Acetohydroxy acid isomeroreductase

0 OH CM3 0 NADPH CM3 OH

H3C-C -C-C'< 'IHO-.-C/< PH C CIHCH3 CH3INADIC3CM M3 0-R NADP CR3 H

a -aceto lactate ,6- hydroxy -a - ketoisova lerate

a, e -dihydroxy-isova lerate

CH3 0 NADPHm 11 ,40 i-

HO-C-- C-C C< _ lw

CH3 0 NADPI a -ketopontoa te

H CH3 OMII &0

HO C C C C

H CH3 H

pontoate

Ketopantoic acid reductaseor

Acetohydroxy acid isomeroreductaseFIG. 2. Comparison of acetohydroxy acid isomeroreductase and ketopantoic acid reductase reactions.

The material for auxanographic studies was ob-tained by incubating 9 U of acetohydroxy acidisomeroreductase for 24 h at 37°C in reaction mixture(20 ml) that contained the following: potassium phos-phate (pH 7.5), 4,000 ,mol; MgCl2, 80 R±mol; NADP 10,umol; glucose 6-phosphate dehydrogenase (Sigma) 16U; potassium a-ketopantoate (pH 7.0), 200 ,umol; andglucose 6-phosphate, 200 ,umol. An additional 200,umol of potassium a-ketopantoate (pH 7.0) and 200,mol of glucose 6-phosphate were added 12 h after thereaction was initiated. At the end of the reactionperiod, the reaction mixture was centrifuged for 10min to remove debris. The supernatant fluid wasdecanted, titrated to pH 5.0 with hydrochloric acid,and allowed to stand at room temperature for 1 h. Thesupernatant fluid was then adjusted to pH 7.0 withsodium hydroxide and extracted with two 10-ml por-tions of ethyl acetate. The ethyl acetate layers werecarefully removed and rotary evaporated to dryness.The resulting material was dissolved in 2 ml of deion-ized water and filter sterilized. Samples (0.2 ml) of thismaterial were spread onto isoleucine-valine agar medi-um. This medium was then streaked with cultures ofpanC2 (pantothenate auxotroph), DU6543 (pantoaterequirer), DU6544 (ketopantoate auxotroph), or ani1vC+ derivative of DU7410 (ketopantoate auxotroph)to determine whether pantoate was present.The material for chromatographic analysis was pre-

pared by incubating 7.5 U of acetohydroxy acid isom-eroreductase for 30 h at room temperature with asolution (2 ml) containing the following: potassiumphosphate (pH 7.5), 400 ,mol; potassium a-ketopan-toate (pH 7.0), 8 ±mol; NADPH, 16 Rmol; MgCI2, 8,umol. Portions (0.5 ml) of the reaction mixture wereremoved at the beginning and the end of the reactionperiod, titrated to pH 2.0 with hydrochloric acid, andallowed to stand for 30 min to form the lactones ofketopantoate and pantoate. These solutions were thenextracted with chloroform (0.6 ml); the chloroformlayer was then dispensed into 1-dram (ca. 3.7-ml) vialsand rotary evaporated to dryness. The material in thevials was then dissolved in a small volume (1 to 2 ,ul) ofchloroform and analyzed on a Becker gas chromato-

graph (Packard Instrument Co.; model 417) equippedwith a flame ionization detector (set at 250°C) and witha 6-ft (ca. 1.83-m) by 2-mm glass column packed withSP2330 (100-200 mesh) Supelcoport (Supelco, Inc.,Bellafonte, Pa.). The column temperature was main-tained at 225°C, and nitrogen was used as the carrier.Air and hydrogen gas rates were adjusted for optimalflame ionization detection. Solutions of authentic a-ketopantoate and D-pantoate were also titrated to pH2.0, extracted into chloroform, evaporated to dryness,and used as standards. Complete resolution of keto-pantoyl lactone and D-pantoyl lactone standards wasobtained; ketopantoyl lactone eluted at 2.71 min, andD-pantoyl lactone eluted at 2.86 min after sampleinjection under the chromatographic conditions de-scribed above. (Ketopantoyl lactone and pantoyl lac-tone from reaction mixtures were identified by com-parison of their retention times to those of thestandards.)

Bacterial strains and culture media. All strains de-scribed in this paper are derivatives of S. typhimuriumLT2 (Table 1). The minimal medium used in all experi-ments was that of Davis and Mingioli (9) which wasmodified by raising the concentration of glucose to0.5% and omitting the citrate; the concentration ofpotassium phosphate and ammonium sulfate was dou-bled in medium to be used for growth experiments orin the 250-ml batch cultures used for the preparation ofcell-free extracts.

Mutagenesis and analysis of nutritional requirements.S. typhimurium strain DU201 (ilvC8) was mutagenizedwith N-methyl-N'-nitro-N-nitrosoguanidine and en-riched for pantothenic acid auxotrophs by the generalmethods of Miller (18). Ampicillin (20 ,ug/ml) was usedto counterselect prototrophs in the first two cycles,and D-cycloserine (2 ,umol/ml) was used in the thirdcycle. Isolates were initially characterized by streak-ing a saline suspension of a single colony ontoisoleucine (50 jig/ml)-valine (100 xg/ml) agar minimalmedium supplemented with either 0.2 mM potassiuma-ketopantoate (25.6 ,ug/ml), 0.2 mM potassium D-pantoate (26 ,ug/ml), or calcium D-pantothenate (20jig/ml) and onto unsupplemented isoleucine-valine

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TABLE 1. Strain listStrain Genotype Growth requirements Source

Wild typeilvC8ilvC8 panES4iIvC8 panES6ilvC8 panES8iIvC8 panE61idvC+ panES4iIvC+ panES6iIvC+ panES8iIvC+ panE54 sup6idvC+ panES4 sup45iIvC+ panES4AilvGE605 ilvBl (zia-1::TNIO)

ilvC+ panES4AilvGE605 iIvBI (zia-I::TNIO) sup601

ilvC+ panES4AilvGE605 ilvBl (zia-I::TnJO) sup6O3

ilvC+ panES4AilvGE605 iIvBI (zia-I::TnlO) sup6O4

ilvC+ panES4 ilvG+ilvB sup601

iIvC+ panES4 i1vG+ilvB+ sup6O3

ilvC+ panE54 ilvG+ilvB+ sup6O4

panC2panB41 iIvC8panD2 ilvC8panDI23 ilvC8

NoneIsoleucine and valineIsoleucine, valine, and pantothenateIsoleucine, valine, and pantothenateIsoleucine, valine, and pantothenateIsoleucine, valine, and pantothenatea-Ketopantoate, pantoate, or pantothenateoa-Ketopantoate, pantoate, or pantothenatea-Ketopantoate, pantoate, or pantothenateNone (stimulated by valine)Valine or a-ketoisovalerateLeucine, isoleucine, valine, and

pantothenate

Leucine, isoleucine, and valine



Valine

None

None (stimulated by valine)

PantothenateIsoleucine, valine, and a-ketopantoateIsoleucine, valine, and P-alanineIsoleucine, valine, and P-alanine

a MNNG, N-Methyl-N'-nitro-N-nitrosoguanidine.

Laboratory collectionF. ArmstrongMNNG' mutagenesis of DU201MNNG mutagenesis of DU201MNNG mutagenesis of DU201MNNG mutagenesis of DU201P22 transduction of DU6543P22 transduction of DU6563P22 transduction of DU6583Pan' revertant of DU6544Pan' revertant of DU6544See text

Pan' revertant of DU6540



See text

See text

See text

K. SandersonMNNG mutagenesis of DU201UV mutagenesis of DU201MNNG mutagenesis of DU201

agar medium. Pantothenate auxotrophs that grew onD-pantoate- but not a-ketopantoate-supplemented me-dium were selected for further analysis. Isolates withphenotypes corresponding to panB, panC, and panDmutant strains were also obtained by this procedure.Growth rates were determined by measuring the

increase in the optical density of log-phase cultureswith a Klett-Summerson colorimeter equipped with a

no. 42 (blue) filter; growth rates are expressed asexponential growth rate constants (generations perhour). Starter cultures for these experiments weregrown overnight in nutrient broth at 37°C. To minimizenutrient carryover, cells were washed once or twice insaline and diluted 10-fold into minimal Davis-Mingiolimedium. A 0.1-mi sample of the washed, diluted cellswas used to inoculate 5 ml of medium. Cultures wereincubated in sidearm flasks at 37°C with shaking.Metabolic intermediates and end products were addedto liquid media at the following concentrations: sodi-um a-ketoisovalerate, 50 ,ug/ml; potassium a-ketopan-toate and potassium D-pantoate, 40 jLg/ml; calciumD-pantothenate, 20 jLg/ml, L-methionine, 50 ,ug/ml; L-leucine, 100 ,ug/ml; L-lysine, 20 iLg/ml; diaminopimelicacid (mixture of LL, DD, and meso isomers), 20 ,ug/ml;and &-aminolevulinic acid, 20 ,ug/ml.

Strain construction and selection of Pan+ revertants.P22-mediated transductions were performed as previ-

ously described (17). Strains DU6543 (panES43 ilvC8)and DU6563 (panE563 ilvC8) were transduced to ilvC+with HT105/4 phage (22) grown on S. typhimuriumLT2 by plating transductants onto pantothenate-sup-plemented medium. Pan' revertants of panE mutantstrains (e.g., DU6544) were obtained by diluting over-night nutrient broth cultures into saline, plating 106 to108 cells onto minimal or leucine-isoleucine-valineagar medium, and incubating at 37°C for 48 h. Thelinkage of panE suppressor mutations to ilvC wasdetermined by transducing strain DU6543 with phagegrown on a suppressor strain (e.g., DU6545) andselecting ilvC+ transductants on pantothenate-supple-mented medium. Cotransduction of the suppressormutation into the recipient strain was determined byscoring the recipient on minimal or valine agar medi-um.

RESULTS

Reduction of a-ketopantoate by acetohydroxyacid isomeroreductase. A preparation of isomer-oreductase that was judged to be greater than95% pure by electrophoretic analysis under de-naturing conditions was used to determine kinet-ic parameters with a-ketopantoate as substrate;the Km was 0.25 mM and the Vmax was 0.136

LT2DU201DU6543DU6563DU6583DU6611DU6544DU6564DU6584DU6545DU6546DU6540

DU6601

DU6603

DU6604

DU6801

DU6803

DU6804

PanCDU7410DU8020DU8123

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pLmol/min per mg. The apparent affinity of theenzyme for a-ketopantoate was similar to thosefor the substrates a-acetolactate (Ki,, 0.30 mM)and a-acetohydroxybutyrate (Ki,m 0.79 mM).The rate of reduction of a-ketopantoate, howev-er, was approximately 20-fold lower than that ofa-acetolactate (Vmax = 2.45 ,umol/min per mg).The reduction of a-ketopantoate to form pan-toate specifically required NADPH; NADH wasinactive. Arfin and Umbarger (1) have shownthat Mg2+ is required for the reduction of a-keto-,-hydroxyisovalerate by purified isomero-reductase. We found that Mg2+ was required forthe reduction of a-ketopantoate by isomerore-ductase. Arfin and Umbarger (1) have alsoshown that the isomerized intermediate of a-acetolactate, a-keto-,-hydroxyisovalerate, isreduced by isomeroreductase, but, as with a-ketopantoate, the rate of reduction is less (10-fold) than with a-acetolactate. We showed bystandard kinetic analysis that a-ketopantoateappears to be a competitive inhibitor of theconversion of a-aceto-a-hydroxybutyrate toa,3-dihydroxy-,-methylvalerate. The Ki com-puted from conventional Lineweaver-Burkeplots was approximately 0.4 mM, which wasclose to the Km value obtained with a-ketopan-toate.Formation of pantoic acid from a-ketopantoic

acid in the isomeroreductase-catalyzed reaction.The reduced product of the isomeroreductasereaction with a-ketopantoate as substrate wasshown to be pantoic acid (pantoyl lactone) bytwo criteria. (i) Material extracted from isomero-reductase reaction mixtures could support thegrowth of a-ketopantoate-pantoate auxotrophs,DU6544 (panE54) and DU7410 (panB41), as wellas a strict pantoate auxotroph, DU6543 (panE54ilvC8); this material could not support thegrowth of a panC mutant. (ii) Material extractedfrom reaction mixtures was analyzed by gas-liquid chromatography. This material containedtwo substances that had the same retentiontimes as ketopantoyl lactone and pantoyl lactonestandards. The peak corresponding to ketopan-toyl lactone (retention time, 2.71 min) de-creased, and the pantoyl lactone peak (retentiontime, 3.15 min) increased during the course ofthe reaction.

Ketopantoic acid reductase activity in crudeextracts of S. typhimurium. Crude extracts of S.typhimurium containing isomeroreductase cata-lyzed the reduction of a-ketopantoate underconditions that are optimal for the activity of thepurified enzyme (pH 7.5, 4 mM Mg2+). Extractsof ilvC mutant strains would not catalyze thereduction of a-ketopantoate under these condi-tions. Crude extracts of ilvC and ilvC+ strainscontained an additional ketopantoic acid reduc-tase activity that is optimal at pH 5.0, is Mg2+

independent, and requires NADPH. This activi-ty probably is homologous with an activity inextracts of E. coli which was previously de-scribed (H. L. King, Jr., and D. R. Wilken, Fed.Proc. abstr. 476, 1972). Evidence is presentedbelow to suggest that this activity functions as aketopantoic acid reductase.

Selection and identification of S. typhimuriumstrains defective in ketopantoic acid reductase. S.typhimurium strain DU201 (ilvC8) was mutagen-ized with N-methyl-N'-nitro-N-nitrosoguanidine(18) and enriched for pantothenic acid auxo-trophs by the ampicillin selection technique (18).Four different phenotypic classes of pantothe-nate-requiring auxotrophs were obtained. Threeof these corresponded to mutants already de-scribed: panB mutants, which require either a-ketopantoate, pantoate, or pantothenate; panCmutants, which require pantothenate; and panDmutants, which require P-alanine or pantothe-nate. Mutant strains in the remaining class,which we designate panE, were distinguished bytheir ability to grow in minimal medium supple-mented with pantoate or pantothenate, but notin minimal medium supplemented with a-keto-pantoate or P-alanine. On the basis of this phe-notype, strains DU6543, DU6563, DU6583, andDU6611 were characterized as mutants thatwere unable to convert a-ketopantoate to pan-toate. The growth rates of DU6543 and DU6563in the presence of isoleucine and valine andother compounds are shown in Table 2. All fourstrains had substantially reduced levels of theMg2+-independent ketopantoic acid reductasedescribed above (3.2 to 12% of the activitypresent in DU201; Table 2, data not shown).Both DU6543 and DU6563 had wild-type levelsof ketopantoate hydroxymethyltransferase (datanot shown). A ketopantoyl lactone reductaseactivity was detected in extracts of S. typhimur-ium LT2 (ilvC8); strains DU6543 and DU6563also had the lactone reductase activity at ap-proximately the same level (2.3 to 3.5 nmol/minper mg of protein). This observation suggeststhat ketopantoyl lactone does not play an impor-tant role in pantothenate biosynthesis.

Role of the a-acetohydroxy acid isomeroreduc-tase in pantothenic acid biosynthesis. To deter-mine the capacity of the isomeroreductase tocatalyze the reduction of a-ketopantoate in vivo,the ilvC+ gene was introduced into three panEilvC8 mutants, DU6543, DU6563, and DU6583,by P22-mediated transduction. The resultingstrains, DU6544, DU6564, and DU6584, all re-tained the requirement for pantoate or pantothe-nate and gained the ability to grow in the pres-ence of a-ketopantoate in the presence orabsence of isoleucine and valine. Growth ratesof DU6544 and DU6564 in the presence ofpantothenic acid intermediates are shown in

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TABLE 2. Exponential growth rates and ketopantoic acid reductase activities of DU201, DU6543, andDU6563

Growth rate (generations/h) in the following media: Ketopantoic acid

IV + lysine reductasebStrain Iva IV + ai-keto- IV + IV + panto- + methionine activitypantoate pantoate thenate + B-ALAC (nmol/min per mg)

DU201 (ilvC8panE+) 1.01 1.06 1.11 1.05 1.13 23.5 (100)dDU6543 (ilvC8 panE54) O' 0.21 0.88 1.05 oe 2.82 (12)DU6563 (ilvC8 panE56) oe 0.19 0.76 0.68 oe 0.74 (3.2)

a IV, Isoleucine-valine medium.b Ketopantoic acid reductase assays were performed at pH 5.5 as described in the text on cells grown in

minimal medium supplemented with pantothenate, isoleucine, and valine.c 8-ALA, 8-Aminolevulinate.d Values within parentheses refer to the percentage of ketopantoic reductase activity in a given strain divided

by the specific activity in DU201.' Growth stopped abruptly after three to four doublings.

Table 3; apparently, the isomeroreductase atuninduced levels is sufficient for pantothenicacid biosynthesis when abundant quantities of a-ketopantoate are present. The phenotype ofpanE mutant strains (DU6544, DU6564, andDU6584) may explain why none has beenidentified. In the presence of a functional iso-meroreductase, these mutants are phenotypical-ly identical to panB mutants (i.e., both requirea-ketopantoate) and could only be distinguishedby the appropriate assays.The requirement of strain DU6544 for panto-

thenate or its precursors could be abrogated byinducing the synthesis of the isomeroreductase.Induction was accomplished by inoculatingDU6544 onto minimal agar medium supplement-ed with -0.5 ,umol of a-aceto-a-hydroxybutyr-ate. Luxurious growth was observed within 24h; no growth was observed in the absence of a-

aceto-a-hydroxybutyrate on minimal medium.This observation indicates that an elevated levelof isomeroreductase allows pantoate to by syn-thesized either (i) by raising the level of a-

ketoisovalerate and concomitantly a-ketopan-toate or (ii) by increasing the rate of conversionof a-ketopantoate to pantoate. The former ex-planation is unlikely in view of the observationthat neither valine (100 ,ug/ml) nor a-ketoisoval-erate (50 ,ug/ml) permitted the growth ofDU6544, DU6564, or DU6584 (Table 3, data notshown for DU6584). Also, it should be notedthat the inducer used in the experiment de-scribed above was the isoleucine intermediate,not the a-ketoisovalerate (valine) intermediate,therefore precluding the possibility that the in-ducer was merely serving as a source of a-ketoisovalerate which might serve to alleviatethe ketopantoate requirement.

Lysine-methionine requirement of panE mu-tants. We have recently demonstrated that thebiosynthesis of pantothenic acid in a S. typhi-murium ilvG mutant strain (DU501) is partiallyblocked and that the concomitant decrease incoenzyme A and succinyl coenzyme A in thisstrain is manifested as a requirement for methio-nine (20). In addition, inhibition of the growth of

TABLE 3. Exponential growth rate of DU6544 and DU6564 in various mediaGrowth rate (generations/h) in the following media:

IV + IV+ IV XStrain a-ke- IV~+ IV + IV + Iysin IV + a-ke- cxK-Lysine

IVa to- p amt + y io-M K- Pan- Panto- Methio- +ov-Keto-pa-tothe- metho e y os- imal topantoate thenate nine methio- Lysine isoval-pantoate nate nine thio- sine valer- toate nine eatetoatentate

DU6544 0" 0.96 1.18 1.30 0 1.09 0 0" 0 0.87 0.90 0.923 0 0.67 0 0"(ilvC,panE54)

DU6564 0" 0.882 1.30 1.03 0 0.674 0 0" 0 1.15 1.18 1.39 0 0.86 0" 0"

panE56),a IV, Isoleucine-valine medium.b No growth was observed within 24 h, growth observed after this period was due to the appearance of Pan'

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wild-type S. typhimurium by inhibitors of panto-thenic acid biosynthesis (e.g., salicylic acid) canbe reversed by the amino acids that requiresuccinyl coenzyme A for their biosynthesis, i.e.,lysine and methionine (20). Methionine, but notlysine, reverses growth inhibition at the mini-mum inhibitory concentration of salicylic acid;at higher concentrations, both lysine and methi-onine are required to reverse inhibition. At thehighest concentrations tested, only pantothenatereverses growth inhibition (20). Since the re-quirement for pantothenate, methionine and ly-sine, or methionine alone reflects the degree towhich the pantothenic acid pathway is blocked,these requirements can be used to assess thesynthesis of pantothenic acid in the panE mutantstrains. Strains DU6543 (ilvC8 panE54) andDU6563 (ilvC8 panE54) did not grow in isoleu-cine-valine medium supplemented with eithermethionine, lysine, or a combination of lysine,methionine, and 8-aminolevulinate (Table 2).The growth of the ilvC+ derivatives, DU6544and DU6563, was not stimulated by either lysineor methionine alone; however, these strainswould grow in medium supplemented with bothlysine and methionine in the presence or ab-sence of isoleucine and valine (Table 3). Thesephenotypes indicate that the isomeroreductaseat uninduced levels synthesizes a sufficientamount of pantoate-pantothenate for all cellularfunctions with the exception of two of the succi-nyl coenzyme A-requiring biosynthetic path-ways.

Suppression of the pantoate requirement ofpanE strains by ilvYC regulatory mutations. panEilvC8 strains (e.g., DU6543, DU6563, andDU6583) reverted to Pan' at a low frequency

(less than 5 revertants per 108 cells) when spreadon isoleucine-valine medium. panE ilvC+ strains(e.g., DU6544 and DU6564) reverted to Pan' ata much greater frequency (-1,000 revertants per108 cells) on the same medium or on minimalmedium. These observations strongly suggestthat most mechanisms of suppression of thepantoate requirement of panE strains require afunctional isomeroreductase. Fourteen Pan' re-vertants of DU6544 were selected from eitherminimal or leucine-isoleucine-valine mediumand assayed for ketopantoic acid reductase andisomeroreductase. None of these isolates hadketopantoic acid reductase activity above that inDU6544; all exhibited elevated levels of isomer-oreductase when grown under the conditions inwhich they were originally selected (data notshown). Two of the revertants that were select-ed on leucine-isoleucine-valine medium,DU6545 and DU6546, were chosen for furtheranalysis. The growth of DU6545 was stimulatedby valine, whereas DU6546 required valine forgrowth; the significance of these phenotypes willbe explained below. Both revertants maintainedelevated isomeroreductase under repressingconditions (Table 4). The levels of threoninedeaminase and acetohydroxy acid synthases Iand II in the revertants approximated thosefound in the parent and responded normally torepression by the branched-chain amino acids(Table 4), therefore suggesting that the apparentconstitutive expression of ilvC was for reasonsother than enhanced internal induction by theacetohydroxy acids. P22-mediated transduction-al analysis of DU6545 and DU6546 showed thatthe suppressing mutation in each of these strainsis tightly linked to ilvC: 96% (48 of 50) in the case

TABLE 4. Specific activities of acetohydroxy acid isomeroreductase, acetohydroxy acid synthases I and II,and threonine deaminase in DU6544 and Pan+ revertants

Sp act (nmol/min per mg)

Growth Acetohydroxy acidStrain conditionsa Acetohydroxy acid synthase Threonineisomeroreductase deaminase

I II

DU6544 (panE54 sup') Pan 56.8 9.50 3.22 132LIVP 19.0 5.42 1.44 50.4

DU6545 (panE54 sup6) Pan 440 4.88 9.37 149LIVP 332 3.08 3.72 62.6

DU6546 (panE54 sup45) Pan 128 11.8 5.80 107LIVP 95.5 3.80 4.75 61.0

DU6801 (panE54 sup601) Pan 231 6.65 6.15 252LIVP 256 5.60 2.49 98



a Pan, Growth medium for preparation of cell extracts was supplemented with pantothenate (20 ,ug/ml); LIVP,growth medium was supplemented with leucine (100 ,ug/ml), isoleucine (50 ,ug/ml), valine (100 ,ug/ml), andpantothenate (20 ,ug/ml).

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of the DU6545 suppressor and 73% (29 of 40) inthe case of the DU6546 suppressor. These datasuggest that the suppressing mutations might liein the regulatory region of ilvC or in ilvY, thepositive regulatory effector for ilvC (4).To eliminate the classes of Pan' revertants

whose suppressing mechanism depends on ele-vation of the a-acetohydroxy acids, a panEstrain which lacked acetohydroxy acid synth-ases I and II was constructed. This strain,DU6540 (panES4 AilvGE6OS ilvBl (zia-l::TnlO)ilvC+), which required leucine, isoleucine, va-line, and pantothenate for growth, yielded 10-fold fewer revertants than did strain DU6543(panE54 ilvG+ ilvE+ ilvB+ ilvC+) when platedonto leucine-isoleucine-valine medium. ThreePan' revertants of DU6540, DU6601, DU6603,and DU6604, were selected for genetic andenzymic analysis. The isomeroreductase level ineach revertant was elevated at least 40-fold overthe level in the parental strain (Table 5) whenrevertants were grown in leucine-isoleucine-va-line medium. The suppressing mutation in eachrevertant was at least 73% linked to ilvC8 by P22transduction (Table 5). Linkage between ilvCand the suppressor mutation was determined bycrossing P22 phage grown on a given suppressorstrain into DU6543 (panE54 ilvC8) and selectingfor ilvC+ transductants on pantothenate medi-um. Cotransduction of the suppressor mutationwas determined by scoring onto either minmalor valine medium, since several ilvB+ ilvG+suppressor strains selected on leucine-isoleu-cine-valine medium were observed to be eithervaline bradytrophs (e.g., DU6545) or valineauxotrophs (e.g., DU6546). This scoring proce-dure revealed that the ilvC+ sup derivative ofDU6543, DU6803 (panE54 sup6O3 ilvB+ ilvG+),was a prototroph and was not stimulated byvaline. DU6801 (panES4 sup601) was a valineauxotroph, and DU6804 (panES4 supO4) was avaline bradytroph. A correlation exits betweenthe phenotype of these derivatives and theirisomeroreductase levels; this correlation will bediscussed below.

The parental suppressor strains, DU6601,DU6603, and DU6604, lack both acetohydroxyacid synthases I and II, and no inducers can bemade in vivo; therefore the regulation of theisomeroreductase was studied in the ilvB+ ilvG+derivatives DU6801, DU6803, and DU6804,where the synthesis of the inducers could beregulated by the branched-chain amino acids.The isomeroreductase levels remained high inthese strains and did not respond to repressingconditions. Threonine deaminase and acetohy-droxy acid synthases I and II were present at thesame levels as in DU6544 (panE54 sup' ilvB+ilvG+) and were repressed by the branched-chain amino acids (Table 4).

Inbibiiom of pantoate synthesis in vivo by a-acetohydoxy acids. Nutritional characterizationof DU6540 (panES4 AilvGE605 ilvBl zia-I::TnlO) revealed that this strain could grow onleucine-isoleucine-valine medium supplementedwith lysine and methionine or supplementedwith methionine alone. This surprising pheno-type indicated that pantothenic acid biosynthe-sis was facilitated in this strain, in spite of thefact that the isomeroreductase is maximally re-pressed (Table 5), pantoate cannot be synthe-sized in any significant quantity in the completeabsence of isomeroreductase, however, sinceDU6543 (panE54 ilvC8) and DU6563 (panE56ilvC8) will not grow on either leucine-isoleucine-valine medium or leucine-isoleucine-valine me-dium supplemented with lysine and methionine.These observations suggested that pantoate syn-thesis by the isomeroreductase was enhanced byraising the ratio of ca-ketopantoate to its compet-itive substrates (a-acetolactate and a-aceto-a-hydroxybutyrate). Four lines of evidence sup-port this view: (i) a-ketopantoate competed witha-aceto-a-hydroxybutyrate for binding to theisomeroreductase (see above); (ii) although 1mM a-aceto-a-hydroxybutyrate stimulated thegrowth of DU6544, 10 mM a-aceto-a-hydroxy-butyrate had no effect; (iii) valine enhanced thephysiological suppression of the pantothenaterequirement of DU6544 by a-aceto-a-hydroxy-

TABLE 5. Specific activities of isomeroreductase and cotransduction of suppressor mutations in DU6540and Pan' revertants

Strain Growth medium' Isomeroreductase sp Elevation (fold) t Cotransduction ofact (nmol/min per mg) sup to ilvC8DU6540 (sup') LIVP 4.60 1 NDbDU6601 (sup601) LIV 188 41 73 (66/90)DU6603 (sup603) LIV 522 113 76 (25/33)DU6604 (sup604) LIV 423 92 85 (39/46)

a LIV, Growth medium was supplemented with leucine (100 1.g/m1), isoleucine (50 .g/ml), valine (100 pg/ml),and pantothenate (20 ,ug/ml); LIV, growth medium was supplemented with leucine (100 ,g/ml), isoleucine (50 .Lg/ml), and valine (100 ,ug/ml).

b ND, Not determined.

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CATALYSIS OF PANTOIC ACID SYNTHESIS

butyrate (data not shown), presumably by inhi-bition of the formation of the a-acetohydroxyacids by acetohydroxy acid synthase I; and (iv)several of the panE suppressor strains (e.g.,DU6545, DU6546, DU6801, and DU6804) wereeither valine bradytrophs or valine auxotrophs.A positive correlation exists between the magni-tude of isomeroreductase production and thedegree of valine independence of the suppressorstrains; this suggests that the higher the level ofisomeroreductase, the less the requirement forblocking the synthesis of the a-acetohydroxyacids.The effect of valine in the facilitation of sup-

pression by a-aceto-a-hydroxybutyrate and inthe stimulation of growth of the suppressorstrains could also be explained by proposing thatadded valine raises the level of a-ketoisovalerateand, hence, enhances the synthesis of a-keto-pantoate. This explanation is unlikely since va-line or a-ketoisovalerate does not stimulate thegrowth of panE strains (e.g., DU6544 andDU6564) in which the synthesis of the a-aceto-hydroxy acids cannot be curtailed by endpro-duct inhibition.

Genetic mapping of the panE locus. An effortwas made to determine whether the mutationswhich confer pantoate auxotrophy are linked tothe panBC gene cluster, located at min 4.5 onthe S. typhimurium LT2 linkage map (21). Thelinkage ofpanE to panC was tested by growingP22 phage on strains DU6543 and DU6563 andtransducing a panC2 strain to pan' in the pres-ence of either a-ketopantoate or pantoate; no a-ketopantoate- or pantoate-requiring transduc-tants were obtained with either donor panEmutant (200 of each scored). The linkage ofpanE to panB was tested by transducingDU6563 (panE56 ilvC8) to pantoate indepen-dence with phage grown on strain DU7410(panB41) in the presence of isoleucine, valine,and a-ketopantoate; no a-ketopantoate-requir-ing transductants were obtained (200 transduc-tants scored).To determine whether the mutations in panE

strains DU6543 DU6563, and DU6611 were lo-cated in the same region, a P22 lysate ofDU6564(panE56 ilvC+) was used to transduce strainsDU6543 (panE54 ilvC8) and DU6611 (panE54ilvC8) to panE+ or to ilvC+. There were approx-imately 30-fold fewer panE+ transductants thanilvC+ transducants in the case of DU6543 and300-fold fewer in the case of DU6611; the fre-quency of panE+ transductants was approxi-mately the same as the frequency of ilvC+tranductants in analogous crosses where thedonor phage was grown on the wild type (panE+ilvC+). These data indicate that the panE muta-tions in strains DU6543, DU6564, and DU6611are clustered in the same region.

In the course of our mutagenesis, we obtainedseveral P-alanine or pantothenate auxotrophs.The mutation conferring the P-alanine require-ment to two genetically distinct mutant strains,DU8020 and DU8123, was found to be linked topanC by P22 transduction: 99.1% in the case ofDU8020 (112 P-alanine auxotrophs out of 113total panC+ transductants), and 97.4% in thecase of DU8123 (112 ,-alanine auxotrophs out of115 total panC+ transductants). These auxo-trophs may represent panD mutants which lackaspartate-1-decarboxylase (7). If so, this findingconflicts with the report by Ortega et al. (19) thatpanD is Icoated at min 89 on the S. typhimuriumlinkage map (21); alternatively, the mutantscould represent a gene that encodes a second,heterologous subunit of asparate-1-decarboxyl-ase. Cronan and co-workers have shown that thepanD gene in E. coli K-12 lies betweenpanB andpanC at min 3 (8).The 1-alanine requirement of DU8020 and

DU8123 provided an additional marker for de-termining the location of panE; however, nolinkage was observed between the two genes inP22-mediated transductions. Time of entry stud-ies using S. typhimurium Hfr donors and panErecipients are being conducted to determine thegeneral location of panE.

DISCUSSIONWe have demonstrated that the acetohydroxy

acid isomeroreductase can catalyze the reduc-tion of a-ketopantoic acid both in vivo and invitro. S. typhimurium strains that lack bothketopantoic acid rductase and the isomeroreduc-tase (e.g., DU6543) strictly require either pan-toate or pantothenate in addition to isoleucineand valine; these strains are strongly blocked inthe conversion of a-ketopantoate to pantoate.The extent to which the isomeroreductase syn-thesizes pantoic acid is reflected in the growthrequirements of ilvC+ derivatives of DU6543and DU6563. DU6544 and DU6564 require ei-ther ketopantoate, pantoate, or pantothenate;the requirement can be removed by inducing thesynthesis of the isomeroreductase. This pheno-type indicates that the isomeroreductase alone issufficient for the biosynthesis of pantoate froma-ketopantoate if (i) the level of a-ketopantoateis raised or (ii) elevated levels of this enzyme arepresent.Induced levels of isomeroreductase increase

the synthesis of pantoate by increasing the rateat which a-ketopantoate is reduced. Elevatedlevels of a-ketoisovalerate do not overcome theblock in pantoate synthesis and do not contrib-ute to suppression of the requirement when theisomeroreductase is induced: (i) the growth ofpanE mutant strains is not stimulated by valineor a-ketoisovalerate; (ii) induction of isomerore-

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ductase (with a-aceto-a-hydroxybutyrate) sup-presses the pantoate requirement in the pres-ence of excess leucine, isoleucine, and valine,i.e., when the synthesis of a-ketoisovalerate islimited; and (iii) genetic suppression by eleva-tion of isomeroreductase can occur in strains inwhich the anabolic formation of a-ketoisovaler-ate is completely blocked.A combination of lysine and methionine sup-

ported the growth of panE mutants. This dualrequirement has been observed in other S. typhi-murium strains in which the biosynthesis ofcoenzyme A or succinyl coenzyme A is cur-tailed. Nonpolarigenic ilvG mutants strainsaccumulate a-ketobutyrate, which inhibits syn-thesis of a-ketopantoate by ketopantoate hy-droxymethyltransferase (20). ilvG mutant strainsthat synthesize an iscleucine-insensitive form ofthreonine deaminase are more severely intoxi-cated owing to the unbridled production of a-ketobutyrate; these strains require methionineand lysine for optimal growth (20). Strains defi-cient in a-ketoglutarate dehydrogenase (sucA)have a growth requirement for succinate or acombination of methionine and lysine (14). Theability of methionine-lysine medium to supportthe growth ofpanE (ilvC+) strains suggests thatthe biosynthesis of pantothenate is partiallyblocked and that the isomeroreductase synthe-sizes an adequate amount of pantoate and coen-zyme A for all cellular functions, with the excep-tion of methionine and lysine biosynthesis.An additional aspect of the in vivo ability of

isomeroreductase to serve as a ketopantoic acidreductase is related to the flux of the a-acetohy-droxy acids through the branched-chain aminoacid biosynthetic pathway. It clearly appearsthat the presence of the a-acetohydroxy acidsprevents the conversion of a-ketopantoate topantoate by the isomeroreductase. In the pres-ence of functional a-acetohydroxy acid syn-thases, high levels of isomeroreductase wererequired to alleviate the reductase deficiency inpanE mutant strains (i.e., the lysine-methioninerequirement), but in a strain (DU6540) where thesynthesis of the acetohydroxy acid intermedi-ates was completely blocked, the residual unin-duced level was able to synthesize pantoate (andcoenzyme A) in quantities sufficient for all thecellular functions, with the exception of methio-nine biosynthesis.

Conditions that limit the supply of leucine orvaline (or both) might also limit the biosynthesisof pantothenic acid. Abrogation of the pantoaterequirement ofpanE mutants by induction of theisomeroreductase suggests a mechanism bywhich pantothenic acid biosynthesis could beregulated by the level of leucine, isoleucine, orvaline. S. typhimurium responds to branched-chain amino acid limitation by derepression of

ilvGEDA and ilvB gene products and by induc-tion of the isomeroreductase when the level ofthe a-acetohydroxy acids increases. This re-sponse would enhance the synthesis of pantoicacid as well as the synthesis of the branched-chain amino acids.

Five spontaneously arising Pan+ revertants ofDU6544 (panE54 ilvC+) exhibited elevated iso-meroreductase. The suppressing mutation ineach strain was linked to ilvC by P22 transduc-tion and may represent alterations in the ilvYgene product or in the ilvC promoter. Selectionfor Pan+ revertants of panE strains that lackboth acetohydroxy acid synthases I and II pro-vides a method for obtaining mutants with al-tered expression of the ilvC gene or ilvY gene.

ACKNOWLEDGMENTSWe thank Barbara Murphy for assistance in selection of

some of the Pan+ revertants. We thank Robert W. Wheat forassistance with the gas-liquid chromatographic analysis. Wealso thank Dorothy Thompson for excellent technical assist-ance and Dayle Wilkins for typing the manuscript.

This investigation was supported by Public Health Servicegrant GM12551 from the National Institute ofGeneral MedicalSciences. D.A.P. is a trainee in the University Program inGenetics supported by Public Health Service grant GM02254from the National Institute of General Medical Sciences.

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3. Blaey, D. L., and R. 0. Burns. 1979. Genetic organizationof the Salmonella typhimurium ilv gene cluster. Mol. Gen.Genet. 177:1-11.

4. BaI y, D. L., and R. 0. Burns. 1980. Gene ilvY ofSalmonella typhimurium. J. Bacteriol. 142:1015-1018.

S. Brown, G. M. 1970. Biosynthesis of pantothenic acid andcoenzyme A, p. 73-80. In M. Florkin and E. H. Stotz(ed.), Comprehensive biochemistry, vol. 21. Elsevier-North Hoiand Publishing Co., Amsterdam.

6. Burn, R. 0. 1971. L-threonine deaminase biosynthetic(SalmoneUa typhimurium). Methods Enzymol. 17B:555-560.

7. Cronan, J. E., Jr. 1980. ,-Alanine synthesis in Escherich-ia coli. J. Bacteriol. 141:1291-1297.

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11. Haler, J. G., C. J. Decedue, G. H. Lgnl, J. A.Reynol, a R. 0. Bum. 1975. The subunit structure ofa-acetohydroxy acid isomeroreductase from Salmonellatyphimurium. J. Biol. Chem. 250:877-882.

12. King, H. L., Jr., R. E. Dyar, and D. R. Wilken. 1974.Ketopantoyl lactone and ketopantoic acid reductases. J.Biol. Chem. 249:-4689-4695.

13. KNJg, H. L., Jr., and D. R. Wllken. 1972. Separation andpreliminary studies on 2-ketopantoyl lactone and 2-keto-pantoic acid reductases of yeast. J. Biol. Chem. 247:4096-4098.

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16. Lowry, 0. M., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275.

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19. Ortega, M. V., A. Cardenas, and D. Ublera. 1975. PanD, anew chromosomal locus of Salmonella typhimurium forthe biosynthesis of j8-alanine. Mol. Gen. Genet. 140:159-164.

20. Primerano, D. A., and R. 0. Burns. 1982. The metabolic

basis for the isoleucine, pantothenate or methionine re-quirement of ilvG strains of Salmonella typhimurium. J.Bacteriol. 150:1202-1211.

21. Sanderson, K. E., and P. E. Hartman. 1978. Linkage mapof Salmonella typhimurium, edition V. Microbiol. Rev.42:471-519.

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decreased transduction abilities. Mol. Gen. Genet.119:75-88.

23. Stormer, F. C., and H. E. Umbarger. 1964. The require-ment for flavin adenine dinucleotide in the formation ofacetolactate by Salmonella typhimurium extracts. Bio-chem. Biophys. Res. Commun. 17:587-592.

24. TeUer, J. H., S. G. Powers, and E. E. Sneil. 1976. Keto-pantoate hydroxymethyltransferase. I. Purification androle in pantothenate biosynthesis. J. Biol. Chem.251:3780-3785.

25. Wllken, D. R., H. L. King, and R. E. Dyar. 1975.Ketopantoic acid and ketopantoyl lactone reductases. J.Biol. Chem. 250:2311-2314.

26. Wilken, D. R., H. L. King, Jr., and R. E. Dyar. 1979.Ketopantoyl lactone reductases. Methods Enzymol.62:209-215.

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