ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1976, p. 224-232Copyright C 1976 American Society for Microbiology

Vol. 9, No. 2Printed in U.SA.

Biogenetic Origin of the D-Isoleucine andN-Methyl-L-Alloisoleucine Residues in the Actinomycins

TAKEHIKO YAJIMA,' KAARIN MASON, AND EDWARD KATZ*

Department of Microbiology, Georgetown University, Schools of Medicine and Dentistry,Washington, D.C. 20007

Received for publication 6 October 1975

Studies with '4C-labeled isoleucine stereoisomers have established thatL-alloisoleucine, D-alloisoleucine, and D-isoleucine may function as precursorsfor the biogenesis of D-isoleucine and N-methyl-L-alloisoleucine residues in acti-nomycin. L-['4C]isoleucine appears to be employed chiefly for n-alloisoleucine(and N-methylisoleucine [?] formation); however, its role in the biosynthesis ofD-isoleucine and N-methylalloisoleucine remains unclear. The potential path-way of biosynthesis of D-isoleucine and N-methyl-L-isoleucine is discussed.

Previous investigations have demonstratedthat synthesis of novel actinomycins as well asenhanced formation of minor components oc-curs during antibiotic production in the pres-ence of one of the four stereoisomers of isoleu-cine (16, 22, 33, 34). i-Isoleucine and N-methyl-L-alloisoleucine were identified as amino acidconstituents of the isoleucine-induced antibi-otics elaborated by various Streptomyces spe-cies (20, 38, 39). The observation that D-isoleu-cine is a constituent of certain monamycin con-geners constituted the first report concerningan exception to the rule of a-epimerization (4,5, 7). Radioisotope experiments clearly re-vealed that L-isoleucine, L-alloisoleucine, andn-alloisoleucine can serve as precursors of thei-isoleucine moiety (11). To clarify the bio-genetic origin of the i-isoleucine and N-methyl-L-alloisoleucine residues in the actinomycins,experiments were carried out employing the'4C-labeled stereoisomers of isoleucine. Theresults of these investigations are presented inthis communication.

MATERIALS AND METHODS

Organisms and conditions of cultivation. Strepto-myces antibioticus (ATCC 14888), Streptomyceschrysomallus (ATCC 11523), and Streptomyces par-vulus (ATCC 12434) were cultivated on slants ofglucose-yeast extract-malt extract agar medium(15). For production of actinomycin, an organismwas grown first in NZ amine medium for 48 h; syn-thesis of actinomycin mixtures was effected subse-quently in glutamic acid-galactose-glucose or fruc-tose-glutamic acid-histidine medium as describedpreviously (19, 21, 23, 39).

Chemicals and reagents. D-Isoleucine and DL-iSO-I Present address: Microbial Chemistry Research Lab-

oratory, Tanabe Seiyaku Company Ltd., Saitama, Japan.

leucine (mixture of four stereoisomers) were pur-chased from Sigma Chemical Co. (St. Louis, Mo.). L-Alloisoleucine was purified from commercial L-al-loisoleucine (Schwarz BioResearch, Orangeburg,N.Y.) or from a mixture of stereoisomeric racematesby the method of Greenstein and Winitz (13). D-Alloisoleucine was kindly supplied by Ted Otani(National Cancer Institute, Bethesda, Md.). L-Iso-leucine was purchased from Calbiochem (San Diego,Calif.). N-methyl-DL-isoleucine was provided byJun'ichi Shoji (Shionogi Research Laboratories,Osaka, Japan), and N-methyl-D-alloisoleucine wasa gift from Tapan Audhya (Dalhousie University,Halifax, Nova Scotia, Canada). N-methyl-DL-valinewas prepared by the method of Cook and Cox (10).All other amino acids were from commercialsources.

i- and L-amino acid oxidases were purchased fromWorthington Biochemicals Corp. (Freehold, N.J.)and the Ross Allen Reptile Institute (Silver Springs,Fla.), respectively. Acylase I (hog kidney) and silicicacid (sil-B-200, 60 to 200 mesh) were from SigmaChemical Co. (St. Louis, Mo.). Silicic acid (100mesh) was procured from Mallinckrodt ChemicalWorks (St. Louis, Mo.).

Radioisotopes. Uniformly labeled L-[P4C]isoleu-cine (312 mCi/mmol) was purchased from Amer-sham-Searle (Arlington Heights, Ill.) and NewEngland Nuclear Corp. (Boston, Mass.), and DL-[3,3',4,5-14C]alloisoleucine (165 mCi/mmol) was agift from Schwarz/Mann BioResearch Inc. (Orange-burg, N.Y.). D- and i_['4C]alloisoleucine were puri-fied from the DL-racemate by the following method.A preliminary experiment (high-voltage electro-phoresis, autoradiography) revealed that the 14C-labeled racemate was contaminated with severalradioactive substances. To purify the radiolabeledamino acid (50 ,uCi), preparative high-voltage elec-trophoresis was carried out. After autoradiography,the area of the paper corresponding to alloisoleucinewas cut out, and the amino acid was eluted withwater by descending chromatography. The eluatewas acidified with 6 N HCI to pH 1.0 and applied to

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a Dowex 50W x 8 column (H+ form, 0.4 by 5 cm).The column was washed with 0.1 N HCl and thenwater until neutral. The amino acid was eluted with1 N NH4OH. After evaporation to dryness, the resi-due was mixed with 5 mg of unlabeled L-alloisoleu-cine and acetylated with 0.05 ml of acetic anhydrideand 1 ml of glacial acetic acid. Acetic acid was re-moved in vacuo after the reaction; the residue wastreated with 0.5 mg of acylase I and 2.5 mg of cobal-tous acetate in a final volume of 2.5 ml (pH 7.5 with1 N NH40H) for 48 h at 38 C. After acidificationwith glacial acetic acid to pH 5.0, protein was re-moved by centrifugation. The volume of the super-natant was reduced to approximately 1 ml in vacuo,and the amino acid solution was applied as a streakto Whatman 3MM paper (46 by 46 cm) and developedwith the solvent mixture (1-butanol-acetic acid-water, 12:3:5) for 16 h. The air-dried paper was sub-jected to autoradiography; two bands were visible onthe X-ray film. Free L-alloisoleucine was elutedfrom lower band (Rf, 0.66) and N-acetyl-D-alloiso-leucine was eluted from the higher band (Rf 0.92)with water. The L-['4C]alloisoleucine solution waspurified further through a Dowex 5OW x 8 column;the NH40H eluate was evaporated to dryness. Theyield of L-alloisoleucine was 10.5 ,uCi (1.96 mg). Thesolution containing acetyl - D- alloisoleucine wasevaporated to dryness, and the residue was refluxedfor 2 h in 1.0 ml of 2 N HCl. The hydrolysate wasevaporated to dryness and purified by Dowex 5OW x8 (H' form) chromatography. The yield of D_['4C]-alloisoleucine was 14.5 ,uCi.

N['4C]isoleucine was prepared from DL-[14C]alloisoleucine in the following manner. A mixtureof DL-['4C]alloisoleucine (4.24 ,uCi, 6.8 x 10" counts/min) and unlabeled L-alloisoleucine (0.4 ,umol) wasadded to each of two flasks of a 48-h-old culture ofS. antibioticus actively synthesizing actinomycin,and the incubation was resumed for 4 h. The com-bined culture filtrates (190 ml) were extracted twotimes with 200 ml of ethyl acetate. The organicphase (containing ['4C]actinomycin) was washedfive times with 100 ml of water. After removal of theethyl acetate by evapor Ltion in vacuo, the actino-mycin mixture (5.06. ,umol, 3.38 x 105 counts/minper ,umol) was hydrolyzed in 6 N HCl, and theamino acids in the hydrolysate were separated byhigh-voltage paper electrophoresis. ['4C]isoleucineon the electropherogram was located by autoradiog-raphy. After elution with water, the '4C-labeledamino acid was further purified by Dowex 5OW x 8ion-exchange chromatography. The D configurationof the amino acid was established with the use of D-and L-amino acid oxidases (23, 25, 30).

Incorporation of '4C-labeled amino acids intothe actinomycins. Experiments were carried outwith a Dubnoff metabolic shaking incubator (Pre-cision Scientific Co., Chicago, Ill.) or with a recip-rocating shaking incubator (New Brunswick Scien-tific Co., New Brunswick, N.J.) at 30 C (23).

Distribution of radioactivity in the constituentamino acids of actinomycin. After incubation, theculture filtrate was separated from the myceliumby filtration. The mycelium was washed with de-ionized water, and the washings were combined

with the culture filtrate. Actinomycin mixtureswere extracted from the aqueous solution withethyl acetate (1:2). The organic phase was washedfour times with an equal volume of water and thenevaporated to dryness under vacuum. The actino-mycins were purified by silicic acid chromatography(23, 35). The antibiotic synthesized by S. anti-bioticus or S. parvulus was hydrolyzed in 6 N HCl asdetailed elsewhere (23). In most experiments, S.chrysomallus mixtures were first separated intoindividual components by descending paper chro-matography and then purified by the procedure ofYajima et al. (39) prior to acid hydrolysis.

Separation of amino acids in hydrolysates wasachieved by high-voltage electrophoresis (23, 38,39). Amino acid standards were also applied tolocate the amino acids. For samples of relativelyhigh specific radioactivity, sections (1 cm long by2.5 cm wide) from a single run were cut from theunstained paper and transferred to counting vialsfor radioactivity measurements in Omnifluor. Forsamples of low specific radioactivity, aliquots of ahydrolysate were applied to Whatman 3MM paperat 0.25-inch (approximately 0.63 cm) intervals (ap-proximately 0.1 ,umol/sample) and subjected tohigh-voltage electrophoresis. After separation, theamino acids were located with ninhydrin (0.02% inacetone) and then eluted from the paper with 75%acetone in water. Eluates were transferred to count-ing vials and evaporated to dryness, and radio-activity was measured in 10 ml of Omnifluor.

For the preparative isolation of amino acids byelectrophoresis (23, 38), samples (containing 0.1,umol of each amino acid) were applied to Whatman3MM paper at 0.25-inch intervals. Amino acidstandards were placed at both sides and the centerof the paper. After electrophoresis, the standardswere located with ninhydrin. The individual aminoacids were eluted with water or 0.01 N HCl bydescending chromatography. One- and two-dimen-sional paper chromatography techniques followedby autoradiography were then employed to identifythe 14C-labeled amino acids.

Radioactive measurements. Radioactive meas-urements were effected in a refrigerated Nuclear-Chicago Mark I liquid scintillation spectrometer.'4C-labeled preparations of actinomycin and aque-ous samples of amino acids were counted in 10 ml ofBray's solution (8). Strips cut from electrophero-grams were measured in Omnifluor. Quenching wascorrected by counting samples in the presence andabsence of a "4C-labeled standard.

Autoradiography was accomplished using Du-Pont Cronex 4 medical X-ray films. "4C-labeledamino acids also were located on paper chromato-grams with an autoradiogram scanner (Packardmodel 7201).

Analytical procedures. The concentration of ac-tinomycin was determined spectrophotometrically(23). Determination of amino acids was accom-plished with the Beckman Spinco automatic aminoacid analyzer (model 120C) (38, 39) or by a mod-ification of the paper chromatographic proceduredescribed by Naftalin (23, 29). Optical configurationof amino acids was established with dialyzed prepa-

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226 YAJIMA, MASON, AND KATZ

rations of L- or 1-amino acid oxidase (23, 25, 30).Radioactive assays were conducted by the method ofKatz and Weissbach (23).Chromatographic procedures and high-voltage

electrophoresis. Silicic acid column chromatog-raphy was employed to purify 14C-labeled actino-mycin preparations (23, 25). Actinomycin in ben-zene (4 ml) was applied to the column (1 by 10 cm)which was washed successively with 200 ml of ben-zene and then 300 ml of a benzene-methanol mix-ture (99:1) to remove impurities. Actinomycin waseluted with 200 ml of benzene-methanol (95:5). Theactinomycins produced by S. chrysomallus wereseparated by descending paper chromatographywith the solvent system 10% sodium o-cresotinate-isopropyl ether-chloroform (3:2:1) and purified asdescribed in an earlier publication (39). Each com-ponent was rechromatographed with the same sol-vent system. Homogeneity of the components wasestablished by circular paper chromatography (19)with the solvent systems: (i) 10% sodium o-cresotinate-n-butyl acetate-dibutyl ether (3:1) and(ii) 10% sodium o-cresotinate-amyl acetate.Amino acids in hydrolysates were separated by

high-voltage electrophoresis (Gilson ManufacturingCo., Middleton, Wis.; 4% formate buffer, pH 1.9,3 h, 3,500 to 4,000 V, 200 mA) or by a combination ofhigh-voltage electrophoresis in one dimension andascending paper chromatography in the second di-mension, using 1-butanol-acetic acid-water (4:1:5,organic phase) (23, 38, 39). In some experiments,amino acids were eluted from electropherogramsand further separated by ascending paper chroma-tography with the butanol-acetic acid-water sys-tem. Unless stated otherwise, amino acids werevisualized with 0.2% ninhydrin in acetone. Homo-geneity of amino acids were routinely checked withthe Beckman Spinco automatic amino acid ana-lyzer.

Purification of L-alloisoleucine from a mixtureof the stereoisomeric racemates. Commercial L-alloisoleucine (200 mg) (alloisoleucine-isoleucine[77.5:22.51) was dissolved in 10 ml of 0.2 M am-monium acetate buffer containing 40% ethanol(pH 3.80) and applied to a column (3 by 90 cm) ofDowex 5OW x 8 (NH4+ form) which had been equil-ibrated previously with the same buffer. The col-umn was developed with the same buffer at 30 ml/h,and 10-ml fractions were collected. L-Alloisoleucinefractions (580 to 680 ml) were combined and evapo-rated to dryness in vacuo. Residual ammonium ace-tate was removed by sublimation at 50 to 60 C for 2to 3 h under reduced pressure. After removal ofammonium acetate, the residue was dissolved in 2ml of hot water, and activated Norit A charcoalwas added. The charcoal was removed by filtrationwhile the solution was still hot, and L-alloisoleucinewas crystallized from hot water-ethanol. The yieldwas 98 mg.

Purification of L-alloisoleucine was achieved alsoby the method of Greenstein and Winitz (13). DL-Isoleucine (98 g) (a mixture of isoleucines and allo-isoleucines) was mixed with 1.2 liters of glacialacetic acid and 180 ml of acetic anhydride, and themixture was maintained at boiling temperature for

ANTIMICROB. AGENTS CHEMOTHER.

2 min. N-acetyl-DL-alloisoleucine was recovered andcrystallized from hot 50% acetic acid. Recrystalliza-tion from hot 50% acetic acid was repeated fourtimes. The yield of crystalline N-acetyl-Drnalloiso-leucine was 38.9 g (60.1% yield, containing 3% ofisoleucine).

Free L-alloisoleucine was obtained by treatmentof N-acetyl-DL-alloisoleucine with an L-amino acid-specific acylase. N-acetyl-DL-alloisoleucine (0.2 mol,34.6 g) and 0.5 g of cobaltous acetate were suspendedin 1.5 liters of water, and the pH was adjusted to7.6 with 3 N NH4OH. Acylase I (10 mg) and waterwere added to make a final volume of 2 liters. Themixture was incubated at 38 C for 18 h. The reac-tion was monitored by means of an analytical aminoacid analyzer until the amount of alloisoleucine be-came constant. L-Alloisoleucine was recovered fromthe mixture and recrystallized from ethanol-water.The yield was 8.6 g (65.6%).

Purity of L-alloisoleucine from both preparationswas checked by high-voltage electrophoresis, paperchromatography, and analytical amino acid ana-lysis. Optical purity was determined by opticalrotatory dispersion and L- and 1-amino acid oxi-dase treatment.

RESULTS

Incorporation of '4C-labeled amino acidsinto actinomycins. The incorporation of 14C-labeled isoleucines into the actinomycins elabo-rated by S. antibioticus is shown in Fig. 1 and 2and Table 1. Short-term experiments revealedthat the most effective precursor was L-alloiso-leucine followed by D-alloisoleucine and 1)-iso-leucine. The kinetics of incorporation of L-isoleucine were atypical, for after a period ofrapid labeling there was a slow, continuousloss of radioactivity (Fig. 1). Identical resultswere obtained in two separate short-term ex-periments with different preparations of L-[14C]isoleucine. At first, the loss was at-tributed to a rapid turnover of a labile form ofradiolabeled actinomycin. However, a quanti-tative recovery of amino acids from actino-mycin was obtained, whereas in two experi-ments 80 to 85% of the 14C label derived fromL-isoleucine was lost after acid hydrolysis andsubsequent Norit A charcoal treatment of thehydrolysate. Separation of the '4C-labeled con-taminant (80 to 85% of the radioactivity, maxi-mum absorption at 315 to 320 nm) was ulti-mately achieved by means of silicic acid chro-matography using benzene-methanol (99:1);actinomycin (15 to 20% of the radioactivity)was eluted from the column with 5% methanolin benzene. The nature of the radiolabeledmaterial has not been investigated further.The results obtained with S. antibioticus, S.

chrysomallus and S. parvulus with ['4C]isoleu-cines as substrates are strikingly similar (Ta-

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bles 1 to 5). Thus, L-[14C]alloisoleucine was

employed by all three organisms, principally asprecursor for synthesis of D-isoleucine andN-methyl-L-alloisoleucine. With S. chrysomal-lus and S. parvulus, a limited interconversionof the amino acid to D-alloisoleucine was ob-served also. Due to the small amount avail-able, D-[14C]isoleucine was employed in only asingle experiment with S. antibioticus, and thedata were essentially the same as those ob-tained with L-['4C]alloisoleucine (Table 3).

E 5u^

0 4E

3

o

t-

.<:10C

E

30 60 90 120 150 180 210 240

Mi n utes

FIG. 1. Incorporation of '4C-labeled amino acidsinto actinomycins synthesized by S. antibioticus dur-ing short-term incubations with L-alloisoleucine(0), D-alloisoleucine (A), and L-isoleucine (0). Theamino acid (40 nmol, 2 x 104 counts/min) was

pipetted into 50-ml Erlenmeyer flasks. Five milli-liters ofa 48-h-old culture ofthe organism previouslycultivated in production medium was introduced.Incubation was carried out in a Dubnoff metabolicshaking incubator at 30 C. Radioactivity incorpo-rated was determined by the method of Katz andWeissbach (23).

Previous investigations had demonstratedthat L-isoleucine could be utilized as precursorfor synthesis of the D-alloisoleucine residues inactinomycins C0 and CQ (1). Unfortunately, the

350

~ 300

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E 250E

200

3O 150 -Alle

, 100

E

-A

1 2 3 4 5 6 7 8 9

DAYS

FIG. 2. Incorporation of '4C-labeled amino acidsinto actinomycins synthesized by S. antibioticusduring long-term incubations. Symbols: 0, L-iSO-leucine (two flasks, 765 gmol, 6.5 x 106 countslmin); 0, L-alloisoleucine (two flasks, 153 1umol,3.14 x 106 counts/min); A, D-alloisoleucine (twoflasks, 76.5 jtmol, 4.7 x 106 countslmin). S. anti-bioticus was incubated in production medium (100ml per flask) for 24 h. Then, the '4C-labeled aminoacid was added, and incubations were continued foran additional 120 to 192 h. Radioactive assays were

carried out by the method of Katz and Weissbach(23).

TABLE 1. Incorporation of ['4C]isoleucine stereoisomers into actinomycins by S. antibioticus

Radioactivepreur- Incubation DurationOrganism Radioactive precur- Amount supplied Iubation ofincuba- Amino acid incorporatedgsor volume tion

nmol counts/min ml min nmol counts/min %S. antibioticus" L-U14C]isoleucine 1,600 1.28 x 107 400 30 87.0 7.0 x 105 5.4

L-['4C]isoleucine 2,000 1.58 x 107 500 240 56.0 4.51 x 105 2.8D-["4C]alloisoleucine 400 1.6 X 106 100 240 26.0 1.05 X 105 6.5L-[U4C]alloisoleucine 400 1.57 X 106 100 120 34.4 1.35 X 103 8.6D-14C]isoleucine 5.4 x 104 5 360 4.8 x 103 8.8

,umol counts/min ml h itmol counts/min %S. antibioticush L-1[4C]isoleucine 1,900 1.27 x 107 500 96 34.0 2.3 X 105 1.8

L-[U4C]isoleucine 765 6.5 X 106 200 120 12.2 1.03 X 105 1.6D-[P4C]alloisoleucine 76.3 4.7 x 106 200 192 6.7 4.13 x 105 8.7L-[14C]alloisoleucine 153 3.14 x 106 200 192 9.9 2.04 x 105 6.5

Short-term experiments. The organism was cultivated for 48 h in production medium, at which time the "IC-labeledradioisotope was added to the culture. Incubation was resumed for the period designated.

b Long-term experiments. The organism was cultivated for 24 h in production medium, the 14C-labeled amino acid was

supplied, and the incubation was resumed as shown.

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228 YAJIMA, MASON, AND KATZ

TABLE 2. Incorporation of ['C]isoleucine stereoisomers into actinomycins by S. chrysomallusand S. parvulus

Radioactive precur- Amount.supplied Incubation DurationOrganism sor voleAmount supplied v of incuba- Amino acid inctrporatedtion

nmol counts/min ml mnin nmol counts/min %S. chrysomallus L-['4C]isoleucine" 460 2.9 x 107 1,000 90 47 2.8 x 106 9.7

D-['4C]alloisoleucine" 5,600 5.6 x 106 300 240 360 3.58 x 105 6.4L-['4C]alloisoleucine' 4,500 4.28 x 106 300 360 240 2.25 x 103 5.3

,umol counts/min ml h /Lmol counts/min %L-[14C]isoleucineh 950 1.62 x 107 500 72 17.1 2.92 x 105 1.8

nmol counts/min ml min nmol counts/min %S. parvulus L-[l4C]isoleucinec 2,000 1.49 x 107 500 60 28.7 2.14 x 105 1.43

L-[ 4C]alloisoleucinec 4,500 4.3 x 106 300 240 1,590 1.51 x 106 35.1MLmol counts/min ml h Amol counts/min %

L-[I4C]isoleucined 1,140 7.6 x 106 300 96 43.9 2.9 x 105 3.8

_Incubationin production medium was for 72 h prior to addition of 4C-labeled amino acid.Incubation in production medium was for 48 h prior to addition of '4C-labeled amino acid.Incubation was for 48 h prior to addition.

d Incubation was for 24 h prior to addition.

TABLE 3. Distribution of '4C-radiolabeled precursor in amino acids derived from actinomycins synthesized byS. antibioticus

Short-term expt (si) Long-term expt (s)Radioisotope precursor Amino acid

1 2 1 2

L-[14C]isoleucine Unknown 1 3 3 6Sarcosine 1 4 6 10Valine 1 4 6 7Isoleucine 7 6 4 8Alloisoleucine 69 48 14 7Threonine 1 10 17 14Proline 2 9 33 39N-methylvaline 1 4 8 7N-methylisoleucine 16 11 7 1N-methylalloisoleucine 1 0 0 0

D-[ '4C]alloisoleucine Unknown 5 0Isoleucine 63 66Alloisoleucine 0 9N-methylalloisoleucine 30 25

L-[ 14C]alloisoleucine Unknown 5 6Isoleucine 56 56N-methylalloisoleucine 36 34

D-['4C]isoleucine Isoleucine 49Alloisoleucine 7N-methylalloisoleucine 43

role of r-alloisoleucine as a substrate was notestablished by Albertini et al. (1). Our resultshave revealed that both S. antibioticus andS. chrysomallus are able to convert n-alloiso-leucine to peptide-bound D-isoleucine and N-methylalloisoleucine. In addition, a significantportion of the '4C-labeled amino acid was in-corporated as n-alloisoleucine into the actino-mycins (C,, Co, and C:,) synthesized by S. chrys-

omallus. When L-['4C]isoleucine was suppliedto this organism, virtually all of the radiolabelwas present in the -alloisoleucine residues ofthe C components. The specific radioactivity ofthe peptide-bound amino acid was equal to thatof actinomycin C2 and it was one-half that ofactinomycin C: (data not shown), confirmingthe results of Albertini et al. (1). By contrast,L-isoleucine was extensively metabolized by

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TABLE 4. Distribution of "4C-radiolabeled precursor in amino acids derived from actinomycins synthesized byS. chrysomallus

Short-term expt (M) Long-termRadioisotope precursor Amino acid expt (C mix-

C, C2 C, E,-E2 ture) (M)

L-[14C]isoleucine Isoleucine 2 3 2 11Alloisoleucine 98 97 98 84Proline 0 0 0 5

D-[ 14C]alloisoleucine Sarcosine 3 0 1 8Isoleucine 25 44 12 21Alloisoleucine 28 38 46 20N-methylalloisoleucine 44 18 41 51

L-['4C]alloisoleucine Sarcosine 2 0 0 2Isoleucine 34 48 32 26Alloisoleucine 14 17 16 12N-methylalloisoleucine 50 34 52 60

TABLE 5. Distribution of "4C-radiolabeled precursor in amino acids derived from actinomycins synthesized byS. parvulus

Short-term expt Long-term exptRadioisotope precursor Amino acid (9,t) (%)S

L-[ 14C]isoleucine Unknown 2 3Sarcosine 1 2Valine 1 6Isoleucine 15 7Alloisoleucine 58 21Threonine 4 12Proline 4 37N-methylvaline 1 6N-methylisoleucine 7 2N-methylalloisoleucine 6 2

L-['4C]alloisoleucine Isoleucine 50Alloisoleucine 6N-methylalloisoleucine 43

S. antibioticus and S. parvulus with consider-able randomization of the '4C radiolabel notedparticularly in the long-term experiments. Theshort-term studies did reveal that both orga-nisms utilized the L-enantiomorph for D-alloiso-leucine synthesis. However, the formation ofD-isoleucine and N-methylalloisoleucine fromL-isoleucine was minimal in contrast to ourprevious investigations (20, 38). In fact, theshort-term experiments with L-isoleucine sug-gested that the amino acid may undergomethylation to yield peptide-bound N-methyl-isoleucine.

Identifi'cation of radiolabeled amino acids.In each experiment the relevant "4C-labeledamino acids formed (isoleucine, alloisoleucine,and N-methylalloisoleucine) were isolated andsubjected to autoradiography and radiochro-matographic scanning techniques. It was estab-

lished that the "4C-labeled amino acid co-electrophoresed and co-chromatographed withstandards of isoleucine, alloisoleucine, andN- methylalloisoleucine, respectively. Treat-ment of the ['4C]isoleucine or ['4C]alloiso-leucine derived from hydrolysates with L- and1-amino acid oxidase established that the '4C-labeled compounds possessed the D configura-tion.

DISCUSSIOND-Amino acids have been found as constitu-

ents of microbial cell walls (32), peptidolipids(2), capsules (37), toxins (14), and antibiotics(6, 9). Racemases for various amino acids havebeen observed in microorganisms (28); how-ever, the mechanism of biosynthesis of theD-amino acid residues in most of these peptidemetabolites remains unclear. In some cases,

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both the L- and D-enantiomorphs of an aminoacid can be utilized for synthesis of the pep-tide-bound D-amino acid (18). In other instancesthere is evidence to suggest that only the Lisomer is used for the biogenesis of D-aminoacids in the peptide structures (12, 23). Forexample, Troy (37) recently reported that aparticulate fraction from an encapsulatedstrain of Bacillus licheniformis catalyzed thepolymerization of L-glutamic acid (but not theD isomer) to form a high-molecular-weightpolymer of y-D-glutamic acid. It has also beenobserved that the D-amino acid may actuallyinhibit peptide synthesis (16, 22, 36); such in-hibition is reversed by the L-enantiomorph.

Several hypotheses have been advanced toexplain the biogenetic origin of D-amino acidsin microbial peptides. Mauger (27) has postu-lated that D-amino acids in antibiotics areformed from L-amino acids after incorporationof the latter into stereochemically labile inter-mediates such as cyclic dipeptides (e.g., di-ketopiperazines). Bycroft (9) has proposed thata combined form of a dehydroamino acid de-rived from the corresponding L-amino acidmight be reduced stereospecifically in vivo tothe D isomer during antibiotic formation. Thesehypotheses have not been confirmed experi-mentally. Studies concerned with the cell-freesynthesis of tyrocidine and gramicidin S, how-ever, have revealed that an adenosine 5'-tri-phosphate-dependent racemization of phenyl-alanine occurs while the amino acid is enzymebound in thioester linkage (24).The formation of D-valine, D-alloisoleucine,

and D-isoleucine has been investigated duringactinomycin synthesis in vivo. Radioisotopic(23) and 15N experiments (31) suggest thatL-valine (but not D-valine) is the precursor ofthe D-valine residues in an actinomycin mole-cule and that the inversion of the L-amino acidto its D-enantiomer does not involve a loss ofthe alpha nitrogen. Further, D-valine inhibitsactinomycin synthesis, and this inhibition isreversed by L-valine (16, 22). Albertini et al.(1) similarly showed that L-isoleucine canserve as precursor for D-alloisoleucine synthe-sis, and our present results confirm theseobservations. The role of D-alloisoleucine as aprecursor was not explored prior to the presentinvestigation. Precursor pressure studies haveindicated that the four stereoisomers of iso-leucine may be employed for the production ofactinomycins containing D-isoleucine (and D-alloisoleucine) and N-methyl-L-alloisoleucine(20, 38, 39). The interconversion of the enan-tiomorphic pairs of the isoleucines was postu-lated to involve a-keto acid intermediates (38,39).

ANTIMICROB. AGENTS CHEMOTHER.

The radioisotopic data reported here havedemonstrated that of the isoleucine isomersexamined D-alloisoleucine (S. antibioticus andS. chrysomallus), L-alloisoleucine (S. antibi-oticus, S. chrysomallus, and S. parvulus) andD-isoleucine (S. antibioticus) were readily em-ployed for the biogenesis of the D-isoleucineand N-methylalloisoleucine moieties in theactinomycin peptides. On the other hand, theutilization of these amino acids for D-alloiso-leucine synthesis by S. antibioticus and S.parvulus was considerably less efficient. Allthree organisms can synthesize D-alloisoleucinefrom L-isoleucine. In this respect, our findingswith S. antibioticus and S. parvulus were some-what unexpected, as these organisms normallyelaborate actinomycins containing D-valine(17). Thus, the potential to utilize L-isoleucinefor D-alloisoleucine formation during actino-mycin production is present in these micro-organisms, but it is generally not expressed.Conceivably, this is due to the absence of suffi-cient concentrations of the amino acid in thecell pool as a consequence of stringent regula-tion of its biosynthesis or to its extensivemetabolism.

Studies conducted in our laboratory with L-isoleucine over an extended period of time (16,20, 22, 38, 39) have proved to be inconsistentand puzzling. Consequently, we have been un-able to confirm fully, by means of the "4C-labeled experiments, the results obtained pre-viously employing unlabeled L-isoleucine (16,20, 22, 38). In fact, even the latter studieswith L-isoleucine from different sources haveyielded somewhat conflicting data. For exam-ple, in experiments conducted with L-isoleucinepurified in the laboratory of J. Greenstein(National Institutes of Health [NIH]) (20, 38)or purchased from a commercial source (Cal-biochem) (16, 22), we have observed that newactinomycins were produced (S. antibioticus,and S. chrysomallus). Moreover, quantitativeexamination of acid hydrolysates of the actino-mycins revealed that D-isoleucine and N-methyl-L-alloisoleucine were present at signifi-cantly high levels, replacing D-valine (or D-

alloisoleucine) and N-methylvaline, respec-tively (20, 38). Subsequently, these resultscould not be reproduced using a different iso-leucine preparation from the same commercialsource (unpublished data). Recently, in a simi-lar study with the same L-isoleucine prepara-tion, small amounts of peptide-bound D-isoleu-cine (S. parvulus) and N-methylalloisoleucine(S. chrysomallus) were produced during anti-biotic formation (39). Confirmation of theseresults has been provided by the 14C tracerstudies described here. In all of the previous

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V-ISOLEUCINE AND N-METHYL-L-ALLOISOLEUCINE

investigations in which L-isoleucine was uti-lized for the synthesis of novel biosyntheticactinomycins, N-methylalloisoleucine (but notN-methylisoleucine) and/or D-isoleucine wereidentified as constituent amino acids in hydrol-ysates (20, 38, 39). Preliminary evidence (pre-sented in. Tables 3 and 5) indicate that N-methylisoleucine may also be formed, to alimited extent, from the ['4C]isoleucine .pre-,cursor. Confirmation of this finding is nowunder investigation. In this connection it is ofinterest to note that Audhya and Russellobserved that N-methyl-L-isoleucine and N-methyl-L-alloisoleucine were present in hydrol-ysates of an enniatin A. preparation producedby the fungus Fusarium sambucinum (3).Both the NIH preparation and the com-

mercial preparation used in recent experi-ments were carefully checked for purity bymeans of the analytical amino acid analyzer,high-voltage electrophoresis, paper chromatog-raphy, and the use of i- and D-amino acidoxidase. All of the tests showed satisfactoryhomogeneity and optical purity. The isoleucinefrom commercial sources was examined also byoptical rotatory dispersion and by diastereoiso-meric dipeptide synthesis (26). By contrast,the isoleucine from NIH was provided by thelaboratory of J. Greenstein, where the mostrigorous standards of purity for amino acidswere developed. The authenticity of this com-pound was attested to by former associates ofJ.Greenstein as well as by our own analyses.Thus, both L-isoleucine preparations have ahigh degree of chemical and optical purity.The inconsistent results obtained with "4C-labeled and unlabeled L-isoleucine may be dueto strain variation which could have occurredduring the years of maintenance of the orga-nism. Cloning may provide a more homogene-ous, physiologically constant strain for futureexperiments.

14C radioisotope experiments have estab-lished that -isoleucine, L-alloisoleucine and, toa lesser extent, D-alloisoleucine can serve asprecursor of the D-isoleucine residues in themonamycins (11). Davies et al. (11) proposedthat the a-keto acids most likely function asintermediates which through interconversionby inversion at C-3 could serve as a source ofD-isoleucine. They suggested that -isoleucineunits are incorporated directly into the mon-amycins.Although a-keto acid intermediates un-

doubtedly play a role in the interconversion ofthe isoleucine stereoisomers during actino-mycin synthesis, there is little evidence tosupport the view that the D-amino acids ()-alloisoleucine and n-isoleucine) are used di-

rectly for synthesis of the peptide-bound formof the respective amino acids. Previous investi-gations had revealed that the 1-amino acidsare inhibitory to actinomycin formation (16, 18,22). In the current study, D[14C]alloisoleu-cine in fact generally proved to be a more ef-fective precursor for peptide-bound N-methyl-alloisoleucine and/or D-isoleucine than for D-alloisoleucine (S. antibioticus and S. chrys-omallus). Further, with D_144Clisoleucine (S.antibioticus) we noted an approximately equallabeling of both D-isoleucine and N-methyl-alloisoleucine residues, suggesting that L-alloisoleucine is a common precursor for bothamino acids. However, the possibility that a1-amino acid is used directly for the peptide-bound form of the amino acid cannot be ex-cluded by the data presented here.

Despite a number of unanswered questionsconcerning the metabolic relationships of thebranched-chain amino acids in Streptomyces,we wish to suggest the following tentativescheme to explain the isoleucine interconver-sions:

7!.EN-methyl-L-isoleucineL-isoleucine C

iL 1 D-alloisoleucine

d-a-keto-,3-methylvaleric acid

1 1l-a-keto--methylvaleric acid

L-alloisoleucine --+D-isoleucine,

N-methyl-L-alloisoleucine

The recent studies concerning the adenosine5' - triphosphate - dependent racemization ofphenylalanine during gramicidin S and tyro-cidine formation (24) suggest that perhaps asingle enzyme with rather broad specificitiesfor the L form of the branched-chain aminoacids catalyzes an energy-dependent biosynthe-sis of -amino acids. The mechanism of inver-sion remains to be established.

ACKNOWLEDGMENTSThe investigation was supported by Public Health

Service research grant CA06926 from the National CancerInstitute.

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2. Asselineau, J. i966. The bacterial lipids. Hermann,Paris.

3. Audhya, T. K., and D. W. Russell. 1974. Natural

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