TIIE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 248, No. 1, Issue of January 10, pp. 316-324, 1973

Printed in U.S.A.

Chemistry and Biosynthesis of the Poly(y-D-glutamyl) Capsule

i.n Bacillus licheniformis

II. CHARACTERIZATION AND STRUCTURAL PROPERTIES OF THE ENZYMATICALLY SYNTHESIZED POLYMER*

(Received for publication, JuIy 27, 1972)

FREDERIC A. TROY

Fro?n th.e Departme& of Biological Chemisby, University of California School of Medicitte, Davis, California

956i 6

SUMMARY

Results of chemical, physical, and enzymatic analyses have established that the product of a cell envelope-asso- ciated polyglutamyl synthetase, isolated from an encapsu- lated strain of Bacillus licheniformis ATCC 994541, is p01y(y- D-glUtamiC acid).

Proof that the optical configuration of the glutamyl residues in the enzymatically synthesized polymer and the native capsular polymer is D, has been provided by (a) chromato- graphic separation of diastereoisomeric dipeptides obtained by derivatization with L-leucine-IV-carboxyanhydride, and (b) gas-liquid chromatographic separation of N-trifluoro- acetyl-L-prolylglutamyl dipeptide methyl esters.

The amide linkage has been shown to be y by (u) kinetics of mild acid catalyzed hydrolysis and (b) LiBH4 reduction of the methyl ester of the polymer followed by acid hydrolysis which yielded y-amino-6-hydroxyvaleric acid. Identical treatment of an authentic poly(cy-D-glutamyl) polymer yielded cr-amino-6-hydroxyvaleric acid. Neither the enzymatically synthesized polymers nor the native capsular polymers synthesized in vivo contained any ester, anhydride, or lactone linkages.

Detailed ultracentrifugal studies have established that the biosynthetic polymers are polydisperse with weight- average molecular weight distribution values ranging from 1.72 x 10” to 3.63 x 105. Similar studies carried out on native 7-D - glutamyl capsular polymers synthesized in vivo showed a much greater degree of polydispersity with apparent weight-average molecular weight distribution values ranging from 8.4 X lo4 to 1.15 x 106.

The biosynthetic polymers are susceptible to enzymatic hydrolysis by a pOly(y-D-glutamyl) depolymerase. This enzyme, which readily hydrolyzed the native capsular poly- mer, showed no activity against authentic a-L-glutamy polymers. These data provide additional evidence that the product of the membrane-mediated reaction is structurally

* This study was supported by Grant AI-09352 from the Na- tional Institutes of Health. Preliminary reports of this work

have been presented (1, 2). the series.

Reference 3 is the preceding paper of

identical with the native capsular y-D-glutamyl polymers synthesized in vivo.

Although a considerable amount of effort’ in the past has been directed toward elucidating the structure of the native poly- glutamyl capsular polymers produced by several Bacillus species (4-U), the basic mechanism involved in their biosynthesis re- mains obscure (3). In an attempt to understand this process more fully, studies were initiated with an encapsulated strain of Bacillus licheniformis ATCC 9945A. These studies have shown that a cell envelope particulate fraction contains a polyglutamyl synthetase which catalyzes the polymerization of L-glutamic acid to form a high molecular weight polymer of y-n-glutamate. The results of these studies support t,he contention that the poly(y-o-glutamyl) capsule is synthesized by a sequence of mem- brane-associated enzymatic reactions rather than a mechanism involving the concerted action of a number of soluble trans- aminases, transamidases, or transpeptidases, or both (3). Of fundamental importance to the establishment of this concept is proof that the enzymatically synthesized polymers are structur- ally identical with the native poly(y-n-glutamyl) capsule syn- thesized in vivo.

The present report describes the chemical, physical, and en- zymatic characterization of the enzymatically synthesized and native capsular polymers. The results of these studies estab- lish that the optical configuration of both polymers is D; that the amide linkage is y and that the polymers are polydisperse with an apparent weight-avcragc molecular weight distribution ranging from 1.72 X 10” to 3.63 X 10; for the cnzymatically synthesized polymers and 8.4 x lo4 to 1.15 x IO6 for the native capsular polymers. Furthermore, the biosynthetic polymers are susceptible to enzymatic degradation by a poly(y-n-glu- tamyl) depolymerase which provides additional evidence that they are structurally identical wit.h t.he native poly(y-n-glu- tnmyl) capsule synthesized in vivo.

EXPERIMENTAL PROCEDURE

Organism and Growth ConditionsDetails regarding the growth of the heavily encapsulated strain of B. licheniformis ATCC

316

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,9945A, preparation of the ccl1 envelope particulate fraction and isolation of native capsular y-u-glutamyl polymers synthesized in viva have been dcscribcd (3). Isolation of the enzymatically synthesized capsular polymers used in this study was carried out as described in the preceding paper (3).

Determination oj Opficnl Coniiguration of Glutamic Acid: Syn- thesis and Separafzon of Diastereoisomeric Dipeptides-The optical configuration of glut~amic acid isolated from the enzymatically synthesized polymers, the native polyglutamyl polymers synthe- sized in z&o; chemically symhesizcd a-~- and a-L-polyglutamic acid and a number of authentic glutamyl dipeptidej was deter- mined by chromatogrnphic separation of diastereoisomeric di- peptidrs obtained by derivatization with L-leucine-NCA’ using essentially the procedure described by Manning and Moore (12). Glutamic acid was isolated following hydrolysis of the polymers in 6 N IICl at 110” for 22 hours in evacuated, sealed tubes. The hydroly&es were evaporated to dryness on a ro- tsry evaporator, redissolved in distilled water, and again evapo- rated to dryness. This evaporation procedure was repeated three to four times. For the preparation of dipeptides, samples containing between 5 and 20 pmoles of glutamic acid were dis- solved in 2 ml of 0.45 AI sodium borate, pH 10.2, at 0”. When necessary, the pH was adjusted to 10.2 with 1 M NaOH. A 20% molar excess of L-leucinc-NC4 was added and after 2 min of in- termittent shaking on a Vortex mixer at 4”, the react’ion was stopped by the addition of 0.85 ml of 1 .O N I-ICI. The samples were passed through a Milliporc filter (type HA, 0.45 p filter) and stored frozen, In all of the dsrivatizations reported here, the yield of dipcptide was 90 to 96%. Dipeptide separations were carried out on a Reckman automatic amino acid analyzer ‘(model 121) with a colun~n, 0.9 x 55 cm, packed with Beckman PA35 resin. Elution was with pH 3.10 sodium citrate buffer at a column temperature of 53” and a flow rate of 70 ml per hour. Peak areas were integrated with a 13eckman model 125 Intrgrator. The operational ninhydrin color value for L-leucine-n-glutamic acid (0.59) and L-leuciw-L-gluta.mic acid (0.75) relative to leu- tine (1.00) was used to correct for the true color value of each isomer in samples containing both antipodes as described by Manning and Moore (12).

Synthesis and Gas-Liquid Chromatographic Separation of N- TriJEuoroacetyl-L-prolyl-n-glutanlic Acid and N-Tri$uoroacetyl-L- prolyl-L-glutamic Atid-Derivatization of glutamic acid isolated from the enzymatically synthesized polgglutamyl polymer and the native capsular polymers synthesized in &JO with N-TFAL- prolyl chloride and the subsequent separation of the resultant diastereoisomrrs by 7 gns-liquid chromatography was carried out as follows. The methyl cstrr of glutamic acid, isolated from each polymer by acid hydrolysis as described above, was pre- pared using ethereal diazomcthane. In this procedure, 1.02 to 5.5 pmoles of dry ghnamyl hydrochloride were methylated at 25” by the addition of an excess (3 ml) of freshly prepared ethc- real diazomethane. After brief mixing and the disappearance of the yellow color, an additional 2.0 ml of diazomethane was added and the incubation continued at 25” for an additional 2 hours. The solvent was then evaporated with a flow of dry nitrogen gas. The extent of methylation was quantit,atively determined by fol- lowing the disappearancr of labclcd glutatnic acid and the con- comitant appearance of the labeled methyl ester by paper chro- matography on Whatman No. 3MM in Solvent System 1. The chromatograms were then scamred on a Packard model 7201 radiochromatographic scanner. Quantitative conversion to the

met,hyl esttr was obtained when freshly prepared diazomethane was used.

For the preparation of the N-TFA-L-prolyl derivatives, a lo- fold molar excess of N-TFA-r-prolyl chloride in 1 .O ml of chloro- form was added to each sample of the glut.amyl methyl ester. One milliliter of dichloromethane was then added and the pH adjusted to 9 (pH paper) by the addit’ion of triethylaminc. The reaction mixture was then sealed and heated at 90” for 5 min. After cooling, 1.0 ml of 6 N HCl was added and mixed well on a Vortex shaker. The organic layer was removed, washed three times with distilled water, and dried by shaking with 0.15 g of anhydrous sodium sulfate and finally by passage through a col- umn, 0.5 X 10 cm, of anhydrous sodium sulfate. The column was washed with 3 ml of dichloromethane and the solvent evapo- rated with a stream of dry nitrogen gas. N-TFA-L-prolyl-n- glutamic acid and N-TFA-L-prolyl-n-glutamic acid were resolved on a Varian model 2100 gas-liquid chromatograph (Varian In- struments) equipped with a 6-foot glass column containing 0.5% HI-EFF 2 AI’ on Gas-chrom Q (60 to 80 mesh) (Applied Science Laboratories). The oven temperature was programmed from 140-200”. The 13.6% of the D isomer present in the N-TFA-L- prolyl chloride reagent was corrected for precise quant.itative work. BIternatively, the labeled diastereoisomers were resolved by radio-gas-liquid chromatography on 1.5% OV-210 on Gas- chrom Q at 200”. An Aerograph A-90P gas chromatograph (Wilkins Instrument and Research) was used which was coupled to a Nuclear Chicago Biospan proportional radiation detector (Nuclear Chicago Corp.). Identification of compounds was done by comparing the retention time and position of the radioactive peaks with standards.

Synthesis of y-Amino-F-hydroxyvaleric Bcici-y-Amino-b-hy- drosyvaleric acid was prepa,red by lithium borohydridc reduction of the methyl ester of 5-oxo-2-pyrrolidinc carboxylic acid by a procedure similar to that described by Chibnall et al. (13). Freshly prepared ethereal diazomethanc, 2 ml, at - 10” was added to 8 mg of the dry pprrolidonerarboxylic acid. Following in- cubat,ion at 28” for 2 min, in which there was considerable ef- fervescence, the methyl ester of the pyrrolidonecarboxylic acid became soluble in the ethereal solution. The ethereal diazo- methane was immediately evaporated to dryness with a gentle stream of dry nitrogen gas. The ester was dissolved in 1.0 ml of dry methanol and a lo-fold molar excess of lit,hium borohydride was added. This excess was necessary to insure complete re- duction since lithium borohydride does react with methanol. Other solvents without active hydrogen atoms including tetra- hydrofuran and dichloromethane proved less satisfactory due primarily to the decreased solubility of the ester. The reduced product was dissolved in 1.0 ml of distilled water and deionized by passage through a column, 0.5 x 10 cm, of mixed bed ion ex- change resin (Dowex 5OWX8 and Dowex l-X8). The column was washed with 1.0 ml of distilled water. The reduced ester, which did not adhere to the resin, was adjusted to 6 N HCl and hydrolyzed at 110” for 5 hours. The HCl was removed by repcated evaporation in vacua. The resulting y-amino - 6 - hy- drosyvaleric acid was further purified by preparative thin layer chromatography on Silica Gel F-254 (0.5 mm thickness) plates in phenol-water (100:20, w/v) where it gave a single ninhydrin spot (RF 0.28). The RF for ol-amino-&hydroxyvaleric acid was 0.32. The amino alcohol also gave a single ninhydrin spot (RF = 0.21) on paper chromatography in Solvent System II which is identical with that reported by Chibnall et al. (13) for

1 The abbreviations used are: NCA, i\i-carboxyanhydride; y-amino-F-hydroxyvaleric acid. N-TFA, X-trifluoroacetyl. Ultracenfrifugation Analysis-Ultracentrifugation experiments

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were performed in a Beckman model E analytical ultracentri- fuge equipped with an RTIC temperature unit and automatic speed control. Photographs of the schlieren patterns on glass metallographic plates were measured on a Nikon microcompara- tor. Sedimentation velocity studies were carried out at 25” us- ing the An-D rotor with a 12-mm double sector cell. The po- sition of the boundary at various times was measured from the schlieren diagrams. Determination of the diffusion coeffi- cient for the native capsular polymers was carried out at 20” using a double sector cell with interference window holders and with a capillary synthetic boundary centerpiece. The lower molecular weight species in the polydisperse sample diffused very rapidly leaving a stable Gaussian-shaped boundary from which the diffusion coefficient was determined with the height- area method (14).

For meniscus depletion sedimentation equilibrium runs, a 12- mm double sector cell with interference holders, sapphire win- dows, and an aluminum-filled Epon double sector centerpiece were used. Interference photographs were taken at approxi- mately a-hour intervals as equilibrium was approached to assure that zero concentration of solute at the meniscus was attained. Samples were centrifuged for approximately 45 hours. Measure- ments were recorded when no measurable difference existed be- tween the patterns of two successive photographs. The fringe displacement, [y(r) - ye] at each radial interval was determined as described by Chervenka (15, 16). A plot of the log [y(r) - yO], which is expressed on the figures as lo3 Iog Ay, versus radial displacement was made. Since log [y(r) - yo] is proportional to the concentration of the polyglutamyl polymers, the slope of the plot, d log [y(r) - y&d(?) is directly proportional to the ap- parent molecular weight. Thus, substitution of the slope from this plot into the equation

2 RT M = (1 - op)w2

2.303 (d log [y(r) - yo])

d (4

allows the apparent weight-average molecular weight to be cal- culated (16). The partial specific volume of glutamic acid, 0.66

38,200) and poly(cu-n-glutamic acid) (molecular weight 70,700) were purchased from Miles Laboratories, Inc.

RESULTS AND DISCUSSIOS

Optical Conjfguration of Glutamyl Jioieties-The optical con- figuration of glutamic acid recovered from the enzymatically synthesized capsular polymers and the native capsular polymers synthesized in viva was determined by chromatographic separa- tion of diastereoisomeric dipeptides. A typical separation of L-leucine-n-glutamic acid and L-leucine-L-glutamic acid by ion exchange chromatography on the amino acid analyzer is shown in Fig. 1. As illustrated, this procedure permits the precise and unequivocal determination of the optical configuration of amino acids. Table I summarizes the results of the stereochemical analysis and shows that at least 92.5% of the glutamyl residues isolated from the enzymatically synthesized polymers are of the D configuration. Similarly, the native capsular polymer synthe- sized in viva contains at least 90% of the D isomer. The extent to which the 7 to 10% L isomer contributes as an authentic com- ponent in these polymers is not known with certainty, although in the enzymatically synthesized polymers it may be derived in part from a small amount of endogenous glutsmic acid which is present in the cell envelope fraction (3). Alternatively, the

350 385 420 EFFLUENT, ml

cc per g, was used. For samples containing less than 1 mg ml-r, the Beckman pho-

FIG. 1. Separation of L-leucine-n-glutamic acid and L-leucine-

toelectric scanning system with the high intensity light source at L-glutamic acid by ion exchange chromatography. The diastereo- isomeric dipeptides were obtained by derivatization of authentic

wave length setting of 228 nm was used. Under these condi- samples of L- and n-glutamic acid wit.h L-leucine-NCA as described tions, the optical density of the solution of polyglut.amyl poly- under “Experimental Procedure.” Separation of the dipeptides

mers was a linear function of concentration. was carried out on the Beckman automatic amino acid analyzer

C/rromatogruphy-Paper chromatograms were developed in a (model 121) at pH 3.10.

descending manner using Whatman No. 3MM paper unless otherwise specified. The following solvent systems were used:

TABLE I

I, 1-butanol-acetic acid-water (40:8.8:20); II, l-butanol-acetic Optical conjiguration of glutamic acid in enzymatically synthesized

acid-water (4:1:5); HI, phenol-water (100:20, w/v). Unlabeled and native capsular polymers

I amino acids and amino alcohols were located on paper chromato- grams after treatment with 0.2$Z0 ninhydrin in acetone and heat- Sample

ing at 80-90” for 5 to 10 min. Analytical thin layer chroma- tography was carried out on Silica Gel F-254 plates (0.25 mm thick) which were activated at 100” for 2 hours, cooled, and used Poly(r-n-glutamste). . . immediately. Preparative thin layer chromatography was carried out on the same plates except 0.5 mm thick. Paper Poly (r-n-glutamate). . .

SOWX

Stereochemical analysisa

i D I-

Enzymatically symhe- 92.5 sized

Native capsular polymer 89.5

Chemically synfhesized 92.7 ChemicalIy synthesized 96.9 Chemically synthesized 2.1

7.5

10.6 electrophoresis was carried out on Whatman No. 3MM in pyri- Yoly(ol-n-glutamate)

dinium acetate, pH 3.0, at 60 volts per cm. (mol wt 70,700) _ . 7.2

Jfaterials-Unless otherwise indicated, all chemicals were a-D-Gh-D-Gh.......... 3.1

analytica reagent grade commercial preparations. n-Leucine- r-~-Glu-~-Glu. . . . . 97.8

N-carbosyanhydride, 5-oxo-2-pyrrolidine carboxylic acid, and cr.amino-&hydroxyvaIeric acid were obtained from Cycle Chemi-

(1 Based on chromatographic separation of the diastereoisomeric

cal Co. N-Trifluoroacetyl-n-prolyl chloride was obtained from dipeptides obtained by derivatization of glutamic acid with L- leucine-N-carboxyanhydride as described under “Experimental

Regis Chemical Co. Poly(a-n-glutamic acid) (molecular weight Procedure.”

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L isomer may result from rncemieation during hydrolysis. In ,order to determine thr contribution by this latter process, the ,optical configuration of glutamic acid in a chemicaally synthesized ,cr-u-glutamyl polytner and chemically synthesized glutamyl di- peptides was determined. ,4s shown in Table I, an cu-n-glutamyl polymer of 70,700 molecular weight yielded 7.2% of the L isomer when analyzed under identical conditions. Thus, assuming ,optical purity of this polymer, as much as 7.2% racemization may occur during acid-catalyzed hydrolysis of a-linked glutamyl polymers. Only 2 to 3% raccmization occurred, however, with the chemically synthesized Y-L- and a-n-glutamyl dipeptides. The difficulties in establishing the precise degree of racemizat.ion occurring during acid hydrolysis has been stressed by Manning and Moore (12). Wiltshire (17) also reported that the 6.6% n-glutamic acid found in myoglobin may be attributed to race- mization during acid hydrolysis.

Confirmation that at least 90% of the glutamyl residues from the enzymatically synthesized polymers were of the D eonfigura- tion was obtained by gas-liquid chromatographic separation of the N-TFA-L-prolyl derivatives. This procedure, initially de- scribed by Halpern and Westley (18, 19), permits an accurate ,determination of the optical configuration of amino acids by coupling the methyl ester of the amino acid to N-TFA-L-prolyl chloride. This resolving agent introduces a second center of asymmetry which is necessary for separation of the isomers. The reaction is quantitative and no racemization occurs. Since this resolving agent is volatile, it permits the derivatized diastereo- isomers, either N-TFB-L-proline-n-glutamic acid or N-TFA-L- proline-L-glutamic arid to be resolved by gas-liquid chromatog- raphy as shown in Fig. 2. The panel on the left shows the elu- tion profile obtained wit’h authentic Q4C]glutamic acid and the center panel, the profile of @C]glutamic acid. The right-ha& panel shows the profile of the derivative obtained from the en- zymat’ically synthesized polytners and clearly shows that greater

! R~~zl3.2 min

‘\

L I 5 IO 15 20 0 5 IO 15 200 5 IO 15 20

TIME. min.

FIG. 2. Gas-liquid chromatographic separation of N-TFA-L- proline-n-glutamic acid and LV-TFA-L-proline-L-glutamic acid. Authentic samples of L-[WJglutamic acid (A), D-[%]glutamic acid (B), and the [14C]glutamic acid isolated from the enzymati- tally synthesized polymers (C) were derivatized with N-TFA-L- prolyl chloride. The resultant diastereoisomeric dipeptides, either NV-TFA-L-proline-D-glutamic acid or N-TFA-L-proline-L- glutamic acid, were resolved by radio-gas-liquid chromatography as described under “Experimental Procedure.”

319

than 90% of the radioactivity and mass, which are coincident, is of the D configuration.

Defermination of Amide Linkage-The amide linkage in both the enzymatically synthesized polyglutamyl polymers and the native capsular polymers synthesized in z&o was determined to be y by its susceptibility to acid-catalyzed hydrolysis. As shown in Fig. 3, radiochromatographic and ninhydrin analysis of the hydrolysates following treatment with 2 N HCl at 110” for 30 min showed a loss of radioactivity in the polymer with a con- comitant and stoichiometric increase of radioactivity in a series of glutamyl oligopeptides with varying degrees of polymeriza- tion (Frame 2). After 60 min of hydrolysis (Frame d), essentially all of the radioactivity was present as glutamylglutamylglutamic, glutamylglutamic, and glutamic acid while after only 120 min of hydrolysis (Frame 5), the oligopeptides had been completely hy- drolyzed to glutamic acid. The radiochromatographic profile represents the enzymatically synthesized polymers while the dashed lines represent the ninhydrin-reacting material obtained from the native capsular polymers synthesized in vivo. The oligopeptides from both polymers were of the homologous series of y-n-glutamyl oligopeptides since their chromatographic mo- bility fell on a linear plot of ln[(l/RF) - l] versus assumed de- gree of polymerization as described by Pardee (20). This la- bility of glutamyl amide linkages to acid hydrolysis is similar to the acid lability of poly(y-n-glutamic acid) reported by Waley (9) and is in marked contrast to the stability shown by a-amide glutamyl linkages. Thus, hydrolysis of authentic (chemically synthesized) pOly(a-D-ghtamk acid) (molecular weight 70,700) under identical conditions showed no ninhydrin-reactive prod-

I

2

t P3 D 0

4

5

0 Time

30 min I

45 min I

120 min

FIG. 3. Kinetics of acid hydrolysis of the enzymatically synthe- sized and native r-n-glutamyl capsular polymers. Both polymers were subjected to acid-catalyzed hydrolysis in 2 N HCl at 110”. At the times indicated, aliquots were removed and chromato- graphed in Solvent System I. The chromatograms were scanned for radioactivity (biosynthetic product, radioactivity profile) or st.ained with ninhydrin (native capsular polymer, dashed lines) as described in the text.

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u-AMIDE LINKED POLYGLUTAMIC ACID

0 0

e-o-

(hH2)z

t-0-CHa CH2=N+=N- 1 - (CH,),

Li BH, F&OH + CHrOH

Y I Y I - (CHe),

v I H dHe)z

--N-CH-$-- -N-CH-$- -N-CH-$- H&AH-f-0”

0 0 0 0 a-AMINO-%HYDROXY-

VALERIC ACID y-AMIDE LINKED POLYGLUTAMIC ACID

0 0

b

H (C&h (CHz)z --k-AH-CH,OH H$-LH-CH,OH

0 0

FIG. 4. Scheme illustrating the charact.erization of amide linkages in Dolv&tamic acid. The enavmaticallv svnt,hesixed polymers anb. t& native capsular polymers synthksiled in vivo were methylsted with diazomethane (CH2 = N+ = N-) and re- duced with lit,hinm borohydride (LiBHJ as described under

ucts until 2 hours, a, time after which only a small amount of glutamylglutamic was dctectcd. After 4 hours of hydrolysis, free glutamic acid was dcteetrd while uo glutamyl oligopeptides larger t.han glutamyl~lutarnic were observed. These results are consistent with the gcueral mechauixm for acid-catalyzed hy- drolysis of proteins coutniuing normal a-amide linkages in which the initial products detected are dipeptidex. Furthermore, these data establish that both the enzymatically synthesized polymers and the native capsular polymers produced in viva have identical acid lability profiles cousist,ent with the presence of y-amide linkages.

The apparent lability of the y-amide linkage to acid-catalyzed hydrolysis could possibly be due to the presence of ester-type linkages in the polyglutamyl polymers. In order to rule out this possibility, the reactivity of both polymers wit.h hydroxylamine was tested. Since ucithcr polymer showed any hydroxamate formation following treatment with hydroxylamine and ferric chloride as described by Vogel (21), it is concluded that neither polymer contains an\- ester, anhydride, or lnctone liukagrx and that the observed acid la.bility is due to the presence of y-amide glutamyl linkages.

Confirmatiou of thr presence of ouly y-amide linknges in both polymers was provided by chrmic~nl degradation and subs:ctquent identification of the products. Thus, lithium borohydride re- duction of t,ht> mct,hglatcd polymrrs followed by either acid- or base-catalyzed hydrolysis yielded ouly y-amino-6-hydroxyvaleric acid. ils illustrated by the sequence of reactions showu in Fig. 4, y-amillo-6-h2drosyvalcric: arid cau only arise from au y-amide- linked glutnmyl polymer while au cr-amide-linked glutamyl polymer would yield e-amino-&hydrosyvaleric acid. Indeed, identical t,reat,mcnt of the chemically synthesized oc-u-glutamyl polymer (molecular weight 70,700) yielded only or-amino-s- hydroxyvaleric acid. The two amino alcohols could be sepa- rated by thin layer chronmtography on Silica Gel F-254 plates in

y-AMINO-8-HYDROXY- VALERIC ACID

“Experimental Procedure” for the synthesis of r-amino-&hy- droxyvaleric acid. The reduced polymers were t.hen hydrolyzed in 2 N HCl at 110’ for 4 to 5 hours and the amino alcohol charac- terized

3oor---- I 0 A

0 200

100 a-AMINO-8-HYDROX VALERIC ACID

cm FIG. 5. Proof of r-amide linkage in the enzymatically synt,he-

sized glutamyl polymers. The enzymatica.lly synthesized capsu- lar polymers which were methylated, reduced, and hydrolyzed as described in the legend to Fig. 4 were snbject.ed t,o elect,rophore- sis at pH 3.0 in pyridinium acetat.e at. GO volts per cm as described under “Experimental Procedure.” The distribution of radio- activity was then determined by radiochromatogr~phic scanning and compared to known standards of the two amino alcohols which were subjected to co-electrophoresis and detected by nin- hydrin staining. The unlabeled na.tive capsular polymers syn- thesized in vivo (not shown) were treated in an identical manner. The y-amino-&hydroxyvaleric a.cid from these polymers was detected with ninhydrin (see text for details).

Solvent System III. Under these condit,ions, y-umiuo-&hy- droxyvaleric acid had an Rp 0.28 whilr t,hc cr-amino dcrivat.ive had an RF 0.32. Although this slight tliffelrncc~ in mobility wa,s consistent and reproducible, a more absolute m&hod was rlec- t,rophoretic separa.tion as show11 in Fig. 5. IIt pH 3.0, cY-amino- 6-hydroxyvaleric acid being a dipolar zwit,terion has no elec- trophoretic mobility while y-amino-&hydroxyvaleric acid possesses a net positive charge and, therefore, exhibits a cathodal mobi1it.y. That all of the radi0activit.y from t.hc enzymat,ically synthesized polymers and the ninhydrin-reactive material from the native capsular polymers synt,hesized in viva is associated

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FIG. 6. Sedimentation velocity and schlieren diagram obtained with the native r-n-glutamyl capsular polymers. Ultracentrifu- gation carried out at 25” with 3.1 mg ml-l in 2.5 M NaCl as de- scribed under “Experimental Procedure.” Photograph taken after 35 min at 60,000 rpm; bar angle, 70”. Sample dialyzed against 2.5 M NaCl for 96 hours and the dialysate of the sample solution was used in the reference cell.

0.4oc

0.3oc

3 - s-

(I)

0.200

0.100 I I I I I I I I L I.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.1

CONCENTRATION, mg ml-’

FIG. 7. Concentration dependence of the sedimentation coeffi- cient of native capsular y-n-glutamyl polymers in 2.5 M NaCl.

with y-amino-&hydroxyvaleric acid provides unequivocal proof for the sole involvement of y-amide glutamyl linkages in both polymers. A standard of y-amino-&hydroxyvaleric acid was prepared by LiBH4 reduction of the methyl ester of 5-0x0-2- pyrrolidine carboxylic acid followed by acid hydrolysis to open the ring as described under “Experimental Procedure.”

Sedimentation Equilibrium and Velocity Studies-Detailed ultracentrifugal studies were carried out on the enzymatically synthesized y-n-glutamyl polymers and the native capsular polymers synthesized in vivo. Both polymers show polydispers- ity. As illustrated in Fig. 6, sedimentation velocity studies on the native capsular polymers at 3 mg ml-l in 2.5 M NaCl showed a single hypersharp boundary. The high concentration of salt was necessary to reduce effectively the viscosity of the solution of polymers essentially to that of the solvent. The

321.

FIG. 8. Determination of the diffusion coefficient for the higher molecular weight component in the native capsular r-n-glutamyl polymers. Initial polymer concentration was 4.1 mg ml-r in 2.5 M NaCI. The top frame (0 time) represents the schlieren diagram after reaching 11,000 mm (8 min after initial solvent laverina). Photographs were taken at 32-min intervals. The stable Ga&- Sian-shaped boundary represented in the bottom franze was taken 96 min after att,ainingspeed and was used to calculate the diffusion coefficient by the height-area method as described under “Experi- mental Procedure.”

sedimentation coefficient was markedly concentration-dependent and the extrapolated s:~,~ value was 7.14 S (Fig. 7). The sedimentation coefficient was calculated using the value of 0.66 as the partial specific volume and correcting for temperature, viscosity, and density of the solvent according to the method of Schachman (22).

An experiment to measure the diffusion coefficient exhibited at early times (Fig. 8) a refractive index gradient curve that de- viated markedly from Gaussian form (top frame). The lower

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2.00 - TABLE II Weight-average molecular weight determination of heavier

components of native capsular y-o-glutamyl polymers

Meniscus denletion sedimentation eauilibrium runs were carried out in the six-channel Yphantis cell at the speed and concentrations indicated. The molecular weight values were determined as described in the text.

-

Rotor speed Concentration

rpm x lo= mvml

4.8 0.30 4.8 0.22 5.2 0.30 5.2 0.22 6.0 0.113 8.0 0.113

I I t I I

l 37.0 D 44.0 :i:g rz %:ZS 36.5 39.0

0 50.0 50.5 45.0 46.0

51.0 5 I .5 52.0

FIG. 9. Determination of the molecular weight (M.W.) of the native capsular r-n-glutamyl polymers. The meniscus depletion sedimentation equilibrium method using the six-channel Yphantis cell at 8000 rpm (25”) was carried out as described under “Experi- mental Procedure.” O---O, poly(y-n-glutamyl) concentration, 6.113 mg ml-1 (inner channel) ; O---O, poly(y-n-glutamyl) con- centration, 0.226 mg ml-l (middle channel); O--O, poly(r-D- glutamyl) concentration, 0.339 mg ml-r (outer channel).

molecular weight species diffused very rapidly leaving a stable Gaussian-shaped boundary with a D~o,~ of 4.61 x lo-* as calcu- lated by the height-area method (14). The deviation from Gaussian form (14) is consistent with the evidence of polydis- persity noted in high speed equilibrium runs at speeds greater than 8000 rpm (see below). The small diffusion coefficient measured for the slowly diffusing constituent is consistent with that obtained for large, elongated polymers, as has b?en reported by Schumaker and Schachman (23).

Experiments to determine the apparent weight-average molec- ular weight distribut,ion of the native capsular y-n-glutamyl polymers, were carried out by meniscus depletion sedimentation equilibrium as described under “Experimental Procedure.” Fig. 9 shows a plot of the log concentration versus radial dis- placement (r2) carried out at 8000 rpm in the six-channel Yphantis cell at three different concentrations. The slope from such a plot at any point is proportional to the apparent weight-average molecular weight at the corresponding radial displacement in the centrifuge cell. These results show two important features: (a) an apparent concentration dependence of molecular weight and (b) a marked deviation from linearity at the highest concentration (0.34 mg ml-i). Both of these fea- tures are consistent with a nonideal or polydisperse solute. That the plot of log concentration versus r2 is experimentally straight at the lower concentration is likely due to the fortuitous cancella- tion of the concentration dependency and heterogeneity on each other a.t this speed (16). The results of this experiment show an apparent weight-average molecular weight distribution between 3.20 x lo5 and 1.02 X 106. Table II summarizes the values for the molecular weight-average of the heavier components of the native capsular polymer at four different speeds and three different concentrations. These results were obtained by sub- tracting the contribution of the lower molecular weight compo- nents from the net displacement against radial position in the six-channel Yphantis cell (24). These results confirm that the

-

TABLE III

Weight -average molecular weight

1.03 x 106 1.03 x 106 1.04 x 106 1.15 x 106 1.13 x 106 1.02 x 106

Weight-average molecular weight distribution and values for lighter components of native capsular r-n-glvtamyl polymers

Meniscus depletion sedimentation equilibrium runs were carried out as described under “Experimental Procedure.” The centrifugal fractionation and molecular weight determination is described in the text.

Rotor speed

*pm x 103 m&?/ml

5.2 0.22 6.8 x lo5 8.0 0.22 8.0 0.34 3.2 X lo5

12.0 1.125 2.1 x 105 20.0 0.22 8.8 x 104 20.0 0.60 8.4 X lo4

Weight -average molecular weight

InitiaI slop@ Bottom of cellb

1.15 x 106 7.26 X lo6

5.8 x 105 3.5 x 106 8.8 x 104 8.4 x 10”

6 Where radial displacement from the meniscus exceeded 30 mm. b Radial displacement nea.r the bottom of the cell.

apparent weight-average molecular weight of the heavier com- ponent is about 1.03 to 1.15 x 106.

Since at higher speeds the effect of polydispersity becomes more obvious (16)) the molecular weight average of the smaller macro- species in the polydisperse solute was determined by increasing the concentration of y-u-glutamyl polymers and increasing the speed of the ultracentrifuge, thereby effecting a centrifugal fractionation, The fringe displacement was measured at equal radial increments and graphical plots of log c (log [y(r) - yo] versus r2 were made as described under “Experimental Pro- cedures.” Results of best straight line visual fits through the initial slope, where displacement from the meniscus exceeded 30 mm, and at a radial distance near the bottom of the cell, are summarized in Table III. These data show an apparent weight- average molecular weight for the smaller molecular species of about 8.4 X 104. Thus, these analyses confirm the polydisperse nature of the native capsular y-n-glutamyl polymers and show an apparent weight-average molecular weight range distribution between 8.4 x lo4 and 1.15 X 106.

This high degree of polydispersity in the native capsular poly- mers may result from an enzyme-catalyzed hydrolysis prior to isolation of the polymers rather than differences in the extent of chain elongation during synthesis since an extracellular, endo- type poly(y-n-glutamyl)depolymerase is detectable at early

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I I I 1 f I I I I

44.0 45.0 46.0 47.0 48.0 49.0 50.0 51.0 52.t

r2

FIG. 10. Determination of the molecular weight (M.W.) of the enzymatically synthesized capsular r-D-glutamyl polymers. The long column meniscus sedimentation equilibrium procedure of Chervenka (15, 16) was carried out on the enzymatically synthe- sized polymers as described in the text,.

stages in the growth cycle of B. lichenijormis (3). Although t,he capsular polymers are isolated from culture filtrates at a time of maximal production, as determined by viscometric measure- ments, it is possible that a considerable amount of degradation has occurred without a noticeable effect on the apparent vis- cosity. Although ultracentrifugal st)udies have not been carried out on the native capsular polymers following extensive enzy- matic hydrolysis, conditions under which the relative viscosity is reduced significantly, radioelectrophoretic analysis has shown that these extensively degraded polymers are electrophoreti- tally mobile. This indicates a considerable decrease in their size, The presence of the poly(y-n-glutamyl)depolymerase in the culture filtrate may also account for the wide variation in molecu- lar weight values reported in the literature for native poly(y-D- glutamyl)capsular polymers isolated from Bacillus species. Thus, Kov&cs and Bruckner (5) reported a molecular weight of 6,400 for the capsular polymer from Bacillus subtilis (B. licheni- formis (3)) while Waley (9) reported a molecular weight of approximately 90,000 for the glutamyl capsule from B. Zicheni- formis. The highest molecular weight reported to date for the glutamyl capsule from Bacillus anthracis is 238,000 (10).

The purified, cnzymatically synthesized y-o-glutamyl poly- mers also showed polydispersity when the molecular weight was determined by the long column meniscus sedimentation equilib- rium procedure of Chcrvenka (15, 16). This method was chosen because of the small amount of ,sample required and the longer column height which enhances the detection of polydispersity. The sample (0.05 ml of 4 mg m-1) in 2.5 M NaCl was run in the capillary-type synthetic boundary -41.Upon centerpiece at 5,600 rpm (25”). The interference pattcrll was recorded and a graphic plot of log concentration verszls r2 was made as previously de- scribed. As shown in Fig. 10, marked deviation from linearity indicates polydispersity. The slope near the meniscus gave an apparent weight-average molecular weight of 1.72 x lo5 while the slope near the bottom of the cell showed an apparent weight- average molecular weight of 3.63 x 10”. Thus, these data show a much narrower molecular weight distribut,ion range than the native capsular polymers which may reflect the absence of the poly(y-n-glutamyl)depolymerase in the cell envelope fraction.

MINUS DEPOLYMERASE

PLUS DEPOLYMERASE

323

FIG. 11. Digestion of enzymatically synthesized capsular 7.D-glutamyl polymers with depolymerase. The purified enzy- matically synthesized polymers were digested with poIy(r-D- glutamyl) depolymerase (pH 6.7, 2 hours) and subjected to elec- trophoresis in pyridinium acetate, pH 6.4, at 60 volts cm-1 for 2 hours. The electrophoretograms were then scanned for radio- activity on the Packard radiochromatographic scanner.

That the weight-average molecular weight. of the smaller com- ponent in the biosynthetic product (1.72 X 105) is larger than the molecular weight of the smaller components in the native capsu- lar polymers (8.4 x 10”) supports this contention. What con- trols the degree of polymerization of glutamyl residues in the membrane-mediated, biosynthetic reaction is completely un- known, but these results show that the molecular weight of the heavier components (3.63 X 10”) is only about 3570 that of the native y-n-glutamyl capsule synthesized in GJO (1.15 x 106). The true size of the undegraded native capsular polymer, how- ever, is not known.

Digestion of Enxymatically Synthesized Polymers with, Capsular Poly(y-o-glutamyl)DepoZymerase-As showi in Fig. 11, the enzymatically synthesized polymers were susceptible to enzy- matic degradation by a poly(y-D-glutamyl) depolymerase iso- lated from the culture filtrate of B. lich.en<formis after 4 days of growth (3). After this period of incubation, the viscosity of the culture filtrate had dropped to nearly that of the solvent indicat- ing that the capsular material synthesized in aiuo at earlier times had been nearly completely hydrolyzed by the extracellular enzyme. Although the depolymcrase has not been extensively purified, it has no activity against aut.hentic poly(a-n-glutamic acid). In addition, it is the only enzyme which we have found which catalyzes the hydrolysis of the l,ol~(y-n-glutaxnyl) capsu- lar polymers. Thus, these results provide confirmator,v evi- dence that the enzymatically synthesized polymers are struc- turally identical with the capsular I)Olv(y-D-gll~tarn~l) polymer synthesized in vivo.

AcknowZedgme&s-I wish to express my appreciation to 11r. W. F. Benisek for his helpful interest in this work. Appreciation is also extended to Mrs. E. Beckman who performed the ultra- centrifugal studies and to Doctors E. G. Krebs and R. S. Criddle for helpful suggestions in the preparation of t,his manuscript.

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Frederic A. TroyOF THE ENZYMATICALLY SYNTHESIZED POLYMER

: II. CHARACTERIZATION AND STRUCTURAL PROPERTIESlicheniformisBacillus-d-glutamyl) Capsule in γChemistry and Biosynthesis of the Poly(

1973, 248:316-324.J. Biol. Chem. 

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