Biosynthesis of the Peptidoglycan of Bacterial Cell Walls · uridine nucleotide substrates in the amidation reaction. Some properties of the reaction are reported. The peptidoglycan

THE JOURNAL OP BIOLOQICAL CEEMKITRY Vol. 243, No.4,IasueofFebruary 25,~~.783-790,1968

Printed in U.S.A.

Biosynthesis of the Peptidoglycan of Bacterial Cell Walls

XI. FORMATION OF THE ISOGLUTAMINE AMIDE GROUP IN THE CELL WALLS OF STAPHYLOCOCCUS AUREUS*

(Received for publication, October 16, 1967)

GERHARD SIEWERT AND JACK L. STROMINGER

From the Department o+f Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 5.9706

SUMMARY

The lipid intermediates in peptidoglycan synthesis in Sfaphylococcus aureus, N-acetyhuuramyl(-pentapeptide)-P- P-lipid and N-acetylglucosaminyl-N-acetyhnuramyl(-pentapeptide)-P-P-lipid, act as acceptors of ammonia in an ATP-dependent reaction in which the ar-carboxyl group of glutamic acid is amidated. Procedures have been de- veloped for the separation of the amide and the amide-free forms of the lipid intermediates, and it has been shown that the amide-free lipid intermediate can substitute for the uridine nucleotide substrates in the amidation reaction. Some properties of the reaction are reported.

The peptidoglycan of bacterial cell walls in many species contains a peptide in which the amino acid sequence is L-Ala- n-Glu-L-Lys-n-Ala. In this peptide the y-carboxyl group of n-glutamic acid is linked to the a-amino group of L-lysine. In most of the organisms which have been examined, the a-carboxyl group of n-glutamic acid is present as an amide; i.e. the second amino acid in the peptide sequence is n-isoglutamine (3-5).

The mechanism of biosynthesis of the peptidoglycan from the two uridine nucleotide substrates, UDP-MurNAcl-pentapeptide and UDP-N-acetylglucosamine, involves the participa- tion of a lipid intermediate, GlcNAc-MurNAc(-pentapeptide)- P-P-lipid (6, 7). The purpose of the present paper is to report that the amide group on the n-glutamic acid is added in an ammonia- and ATP-dependent reaction in which the ammonia acceptor is the lipid intermediate.

METHODS AND MATERIALS

Preparation of Particulate Enzymes from Cells of Staphylococcus aureus Strain H after Lysis with Lysostaphin-In several previous

* This research was supported by research grants from the United States Public Health Service (AI-062471 and t,he National Science Foundation (GB-4552). Pr&ninary’ accounts of this work have appeared (1, 2).

1 The abbreviation used is: MurNAc, N-acetylmuramyl.

studies from this laboratory, the particulate enzyme which catalyzes cell wall synthesis was prepared after disintegration of cells with alumina. In the present study, however, particulate enzyme prepared after disintegration of the cells with the staphy- lolytic enzyme, lysostaphin (8, 9), was used unless otherwise indicated. This preparation has the following advantages. (a) Although the particles catalyze synthesis of the lipid intermediate in peptidoglycan synthesis, they are able to utilize these intermediates only very inefficiently for peptidoglycan synthesis. In studies involving the lipid intermediate, it is advantageous to use a preparation in which these intermediates accumulate and are not utilized. (b) The total yield of lipid intermediates per g of bacterial cells was about 8 times that obtained with particulate enzyme prepared after disintegration of cells with alumina (Table I). The specific activity of formation of lipid intermediates was 2 to 3 times that of the alumina particles.

This enzyme was prepared as follows. Cells of S. aureus strain H (2.0 g, wet weight; harvested at one-fourth maximum growth) which had been washed once with 0.05 M Tris-HCl buffer, pH 7.5, containing 10e3 M MgClz, were suspended in 50 ml of the same buffer. A solution of lysostaphin (0.25 ml; 1 mg per ml; generously given by Dr. Peter Tavormina, Mead Johnson Re- search Laboratories (9)) in 0.05 M Tris-HCl, pH 7.5, and 5 ~1 of DNase solution (1 mg per ml) were added. The progress of lysis was checked by repeatedly measuring the absorbance of the suspension (after dilution with 3 parts of buffer) at 700 rnp. The initial absorbance was about 1.5 and fell to 0.25 at 3) hours. The suspension was then cooled in ice and centrifuged for 10 min in a Sorvall centrifuge at 30,000 x g. The supernatant solution was discarded. The precipitate was washed twice with 0.05 M Tris-HCl, pH 7.5, containing 10-s M MgClz and lop3 M 2-mercaptoethanol, and was finally suspended in 2.5 ml of the same buffer. The concentration of protein was about 10 mg per ml. Note that this preparation sedimented at a much lower g value than was required to sediment the particles obtained after grinding with alumina. In some of the later enzyme preparations, a Mg++ concentration of 10e2 M was used for the lysis of cells. A more compact and still more easily separated particulate enzyme was formed under these circumstances. Its protein concentration was about twice that of the preparation made at lower Mg++ concentrations.

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754 Biosynthesis of the Peptidoglycan of Bacterial Cell Walls. XI Vol. 243, No. 4

TABLE I

Comparison of specific and total activities of particulate enzymes from S. aureus strain H, prepared after grinding cells with

alumina and after lysis of cells with lysostaphin

Specific activity5 Total activity”

5 Millimicromoles of MurNAc-W-pentapeptide incorporated per mg of enzyme protein per hour.

* Millimicromoles of Mur?;Ac-W-pentapeptide per g of bac- teria (wet weight) per hour.

Preparation of Nonamiduted Disaccharide(-W-pentapeptide) - P-P-lipid-Two methods were employed.

Method 1: The composition of the reaction mixture was similar to that given below in the assay of the amidating enzyme, except that it was increased by a 50-fold scale. NH&l was omitted. Purified UDP-MurNAc-W-pentapeptide (ammonia-free; see below) was used, and RNase (final concentration, 40 pg per ml) was added. The mixture was incubated for 1 hour at room temperature and was then extracted with an equal volume (about 2.5 ml) of 1-butanol-6 M pyridinium acetate, pH 4.2 (2: 1) (once), and then with l-butanol (three times). The butanol extracts were back-washed once with water (2.5 ml), combined, and concentrated under vacuum to half the original volume (5 ml). Silicic acid (1 g) and then, slowly, with shaking, 35 ml of chloroform were added. The supernatant solution was separated from the silicic acid and contained less than 0.5% of the total radioactivity of the crude extract. The silicic acid which had the W-lipid intermediate absorbed was suspended in chloroform and placed on a column of 5 g of silicic acid in chloroform as described previously (7). All radioactive material came off as one peak with the solvent, chloroform-methanol (1: 1).

The reaction mixture contained 302 ~1 of 20 mu I-14C-DL- glutamic acid (3.0 mC per mmole), 771 ~1 of 8.2 mu UDP- MurNAc-L-Ala, 600 ~1 of 20 mu L-alanine, 800 ~1 of 20 mu L-lysine, 800 ~1 of 20 mM D-Ala-n-Ala, 1200 ~1 of 50 mu disodium ATP (containing 50 mu KHCOJ, 1200 ~1 of 0.5 M Tris-HCl (pH 8.6), 14 ~1 of 0.5 M MgC12,800 ~1 of crude amino acid-adding enzyme (10) (13 mg of protein per ml), and 600 ~1 of 0.1 M MnC12. L-Alanine was included because the UDP-MurNAc- L-Ala preparation also contained some UDP-MurNAc. The mixture was incubated for 1) hours at 37” and then spotted on Whatman No. 3MM paper (10 bands of 15 cm each). The papers were chromatographed in isobutyric acid-l N NHdOH, 5 :3. The bands having the same mobility as the reference compound, UDP-MurNAc-Ala-Glu-‘4C-Lys-Ala-Ala, were located by radioautography, eluted, and rechromatographed in 95% ethanol-l M ammonium acetate, pH 7.0, 7.5:3). UDP- MurNAc-Ala-W-Glu-Lys-Ala-Ala was eluted. The eluate was evaporated and dissolved in water. Starting with 10.5 X lo6 cpm of n-glutamic acid, 1.46 X lo6 cpm (13.9%) were obtained in the nucleotide product.

The W-lipid intermediate obtained in this way was a mixture of disaccharide(-W-pentapeptide)-P-P-lipid (80 to 90%) and some disaccharide(-W-pentapeptide amide)-P-P-lipid. These two forms could be separated by partition chromatography on Sephadex G-25. A biphasic solvent mixture containing 100 volumes of distilled water, 100 volumes of ethanol, 100 volumes of benzene, and 25 volumes of I-butanol (see below) was equili- brated at 18”, and the phases were separated. Sephadex G-25 (fine, bead form) was suspended in the lower phase. A jacketed column, kept at a constant temperature of 18”, was filled with the suspension to a bed volume of 20 ml (31 x 0.9 cm). The column was first washed with the lower phase (water layer) and then with the upper phase (organic layer) until no more of the lower phase appeared in the effluent. The mixture of ‘%-lipids was concentrated to 50 ~1, diluted with 300 ~1 of the upper phase, and put onto the column. The column was eluted with the upper phase at a flow rate of about 0.3 ml per min. Fractions of 1.5 ml were collected, aliquots of which were counted in a dioxane scintillation solution in a Packard Tri-Carb liquid scintillation spectrometer. The separations obtained are illus- trated below.

PuriJcation of UDP-MurNAc-Ala-Glu-‘4C-Lys-Ala-Ala-In order to remove NH, from UDP-MurNAc-pentapeptide, it was subjected to electrophoresis on prewashed Whatman No. 3MM filter paper for 3 hours in 0.05 M sodium acetate buffer, pH 5.6, at 20 volts per cm. The band of 14C-nucleotide was located by radioautography and eluted with water.

Hydrolysis of W-Lysine-labeled Peptidoglycan by Lysozyme- Peptidoglycan was synthesized from UDP-MurNAc-Ala-Glu- ‘JC-Lys-Ala-Ala, UDP-GlcNAc, and particulate enzyme. The W-labeled components of the incubation mixture were separated by chromatography in isobutyric acid-l M NH40H (5:3) as described previously (6, 12). The origin of the paper chromato- gram, which contained the 14C-peptidoglycan, was cut out and moistened with a mixture of 4 parts of sodium acetate buffer (0.02 M, pH 6.0) and 1 part of lysozyme solution (10 mg per ml). It was incubated in a humid chamber at 37” for 40 to 60 hours. A drop of toluene was added to prevent growth of micro- organisms. After incubation, the paper was dried and eluted with water. Between 70 and 80% of the total radioactivity was eluted. The eluate was spotted on Whatman No. 3MM paper and subjected to electrophoresis as described for the assay of the amidation reaction. A radioautogram of the electrophoretogram was prepared. The nature of the compounds observed is described below.

RESULTS

Method 2: Nonamidated lipid intermediate could also be Assay of Amio!ating Enzyme-The following assay was based obtained by blocking the amidation reaction with deoxycholate on the assumption that the acceptor of the amide group would

(see below). The reaction mixture was the same as used above, except that potassium deoxycholate was added to a final concentration of 0.16% and UDP-MurNAc-14C-pentapeptide which had not been freed of ammonia was used. Incubation, extrac- tion, and silicic acid chromatography were the same as above. For the assay of the amidation reaction, this lipid was used without purification by partition chromatography.

Preparation of UDP-MurNAc-Ala-W-Glu-Lys-Ala-Ala- The compound was prepared by enzymatically adding W-D- glutamic acid, L-lysine, and D-Ala-n-Ala to UDP-MurNAc- L-Ala. The reaction mixture was essentially the same as described by Ito and Strominger (10, 11). The three steps were carried out in one reaction mixture, rather than successively.


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be the lipid intermediate, disaccharide(-pentapeptide)-P-P- lipid, and on the fact that the disaccharide-pentapeptide and disaccharide-pentapeptide amide which could be obtained from the lipid intermediate by mild acid hydrolysis would be readily separable by paper electrophoresis. The incubation mixture contained the following, in a total volume of 60 to 70 ~1: 1.0 ~1 of 0.5 mM 14C-UDP-MurNAc-14C-pentapeptide (14C-lysine- labeled); 1.0 ~1 of 5.0 mu UDP-GlcNAc; 12 ~1 of 0.5 M Tr&HCl buffer, pH 8.5, containing 0.017 M MgC&; 10 ~1 of 0.02 M disodium ATP (neutraliied with KHCOJ; 0.5 ~1 of 0.05 M 2-mercaptoethanol; 3 ~1 of 0.5 M NH&l (adjusted to pH 8.5 with Tris base); and 30 to 40 ~1 of particulate enzyme prepared after treatment of cells of S. uuwus with lysostaphin (10 mg of protein per ml; see “Methods and Materials”).

After incubation for 1 hour at 25”, the lipid intermediates from the reaction mixture were extracted by treatment with a mixture of 1butanol-6 M pyridinium acetate, pH 4.2 (2:l) (90 ~1 first, and then 60 ~1 three times). The extracts were back- washed twice with 60 ~1 of water, combined, and evaporated to dryness.

Mild acid hydrolysis was carried out by adding to the residue 25 ~1 of 2-propanol, 60 ~1 of water, and 10 ~1 of 1.0 N HCl. This solution was incubated for 1 hour at 60°, neutralized with 10 ~1 of 1.0 M KHCOs, and taken to dryness. The residue was dissolved in 60 ~1 of water, and the pH was adjusted to 8.5 to 9.0 by addition of 4 ~1 of 1 M Tris base. Escheriehia coli phosphomonoesterase (2 ~1; 10 mg per ml) was added, and the solution was incubated for 2 hours at 37”. It was found that the acid hydrolysis yielded small amounts of phosphorylated materials, and the treatment with phosphomonoesterase was included to diminish the amount of these materials. Then 4 ~1 of 1 N acetic acid were added. The mixture was spotted on Whatman No. 3MM filter paper and separated by electrophoreais for 5 hours at 20 volts per cm (Beckman-Durrum electrophoresis apparatus) in pyridinium acetate buffer, pH 5.0, containing 0.1 M acetic acid and 0.12 M pyridine. The spots of disaccharide-pentapeptide and disaccharide-pentapeptide amide were located by radioautography, cut out, and counted in a Packard Tri-Carb liquid scintillation counter. Under the above conditions of acid hydrolysis, 3% of disaccharide-14C-pentapeptide was hydrolyzed to W-pentapeptide, which had the same electrophoretic mobility as disaccharide-pentapeptide amide and therefore provided a small blank in the estimation of the amount of disaccharide-W- pentapeptide amide. The separation of disaccharide-pentapeptide and disaccharide-pentapeptide amide in this assay is illus- trated in Fig. 1.

Formation of Amiclated Product Dependent on Prewnce of ATP and Ammonk-When incubations were carried out in the presence of ATP and ammonia (assayed as described above), two major products were formed, corresponding in position to those expected for the amidated and nonamidated disaccharide- pentapeptide. When ATP was omitted, only a single product was formed, corresponding in position to the nonamidated material (Fig. 1). No dependence on ammonia could be observed in these early experiments because of the presence of ammonia in UDP-MurNAc-pentapeptide, which was one of the substrates. When this substrate was freed of ammonia, as described in “Methods and Materials,” and incubation was carried out with particulate enzyme which had been washed twice in buffer, a dependence of the reaction on both ATP (Table 11) and ammonia (Table III) could be demonstrated. Forma-

tion of lipid-P-P-disaccharide-pentapeptide amide was 50 y0 of the maximum at about 4 mu ammonium chloride and 0.5 m~a ATP. L-Glutamine substituted for NH&l (Table IV). How- ever, L-glutamine was inactive in the absence of ,ATP and, since some glutaminase activity could be demonstrated in the particulate enzyme, it is assumed that the activity of L-glutamine was due to its prior hydrolysis to yield ammonia. L-Isoglutamine was inactive (Table IV), and similarly L-asparagine and D- and L-glutamic acid were inactive.

Evidence That Amide Is Substituted on ru-Carbox$ Group of

Phosph0ryiated moterial

Diiride-pentopeptide

Diwride-pentapeptide

Concentration of ATP Disaccharide-pentapeptide in reaction mixture amide formed

Diicchar~$&pe$apeptide

11111 CM c9m 0 109 5,102 0.1 624 8,787 0.2 1,451 10,942 0.4 3,311 11,673 0.8 5,697 10,263 1.6 9,661 10,355 3.2 10,200 9,317

-- _I FIG. 1. Radioautogram of the electrophoretic separation of

disaccharide-pentapeptide and disaccharide-pentapeptide amide. The reaction mixtures are those given in Table XI, tubes 1 and 3.

TABLE II Dependence of formation of disaccharide-pentapeptide amide on

ATP concentration The assay was performed as described in the text with purified,

ammonia-free UDP-MurNAcJ4C-pentapeptide (65,000 cpm per tube) and NH&l (90 mna).

TABLE III Dependence of formation of disaccharide-pentapeptide amide on

NH4Cl concentration The assay was performed as described in the text with purified

UDP-MurNAc-W-pentapeptide (64,000 cpm) and ATP (2.8 mM).

Concentration of NHKl in reaction

mixture Disaccharide-pentapeptide

amide formed

mdf CM CM 0 2,881 21,356 1.6 8,221 22,130 4.0 11,359 21,271 8.0 15,588 19,721

36.4 20,072 I 19,300


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TABLE IV

Amidation of lipid intermediate with NH&l, L-glutamine, ana L-isoglutamine as ammonia Sources

The assay was performed as described in the text, except that. NH&l was added only as indicated in the table. Purified UDP- MurNAcJ*C-pentapeptide (65,090 cpm) was used.

Addition :oncentration

None NH&l

L-Glutamine

L-Isoglutamine

VkM

3.5 7.0 3.5 7.0 3.5 7.0

T Disaccharide- pentapeptide amide formed

cw cpnt 8,141 40,457

27,141 26,039 30,206 23,363 26,593 22,968 28,090 21,905

6,542 42,588 7,955 47,840

TABLE V

Products of hydrazinolysis of UBP-MurNAc-Ala-~4C-Glu-Lys- Ala-Ala and of disaccharide-pentapeptide and disaccharide-

pentapeptide amide obtained from lipid intermediate

Tube 1 contained 24 pl of 0.55 mM UDP-MurNAc-Ala-i4C- Glu-Lys-Ala-Ala (see “Methods and Materials”) and 8.6 ~1 of unlabeled 12 mM UDP-MurNAc-pentapeptide. Tube 2 contained disaccharide-pentapeptide (containing r4C-glutamic acid) prepared from UDP-MurNAc-Ala-14C-Glu-Lys-Ala-Ala in a manner similar to that described under “Assay of Amidating Enzyme” in a 30.fold increased reaction mixture containing deoxycholate (0.16$&). The compound was eluted from the paper. The solution was mixed with 8.6 pl of unlabeled UDP-MurNAc-pentapeptide. Tube 3 contained disaccharide-pentapeptide amide (containing 14C-glutamic acid) prepared as described under “Assay of Amidating Enzyme” in a 30-fold increased reaction mixture, which was eluted from the electrophoresis paper and mixed with 4 ~1 of L-glutamic acid dibenzyl ester HCl (10 mg per ml). All of the samples were taken to dryness in l-ml test tubes and subjected to hydrazinolysis (13). After evaporation of the excess of hydra- zine, the mixtures were dissolved in water, spotted on Whatman No. 3MM filter paper, and separated by electrophoresis (13). The spots of glutamic acid r-hydraaide and glutamic acid dihydrazide were located by radioautography, cut out, and counted in a liquid scintillation spectrometer. For mobilities, see Reference 13.

Tube Glutamic acid Glutamic acid r-hydrazide dihydrazide Dihydrazide

cpnt cpln % 1” 12,920 260 2 26 3,740 49 1 36 1,590 3,270 67

a Obtained from the 1%.uridine nucleotide substrate (amide- free).

b Obtained from the amide-free r4C-lipid intermediate synthesized in the presence of deoxycholate.

c Obtained from the amidated “C-lipid intermediate.

Glutamic Acid-It has been shown that the amide group in the cell wall is substituted on the a-carboxyl group of glutamic acid. The following experiment was carried out to show that this was the position of substitution in the enzymatically synthesized product. The lipid intermediate was synthesized with the use of UDP-MurNAc-14C-pentapeptide labeled with 14C-n-glutamic

acid as the substrate. After acid hydrolysis, the disaccharide- r4C-pentapeptide and disaccharide-r4C-pentapeptide amide in the product were separated by paper electrophoresis as described above. These products were then subjected to hydrazinolysis. The products of hydrazinolysis were separated by paper electrophoresis under conditions in which glutamic acid, glutamic acid cr-hydrazide, glutamic acid y-hydrazide, and glutamic acid cr , y-dihydrazide were all separated (13). r4C-Glutamic acid dihydrazide was formed only from the presumed disaccharide- r4C-pentapeptide amide (Table V).

Formation of Peptidoglycan Product with Enzyme Prepared after Alumina Grinding, and Its Hydrolysis with Lysozyme To Yield Disaccharide-pentapeptide and Disaccharide-pentapeptide Amide-Enzyme prepared after disintegration of the cells with alumina, in contrast to that prepared after lysis with lysostaphin, catalyzes the synthesis of peptidoglycan as well as lipid intermediate. The formation of the peptidoglycan product with this enzyme is stimulated by the presence of ammonia in the reaction mixture (Table VI). This presumably occurs because the disaccharide(-pentapeptide amide)-P-P-lipid is utilized for peptidoglycan synthesis more rapidly than the corresponding nonamidated intermediate.

A peptidoglycan was synthesized enzymatically with this enzyme in the presence of NH, and ATP under conditions which should yield the amidated product. This product was then treated with egg white lysozyme (see “Methods and Materials”) and subjected to paper electrophoresis. Two products were obtained, corresponding in electrophoretic mobility to disaccharide-pentapeptide and disaccharide-pentapeptide amide which

had been obtained by mild acid hydrolysis of the lipid intermediates. These amidated and nonamidated products would not have been seen in earlier studies of the lysozyme degradation products (12), because on paper chromatography in the isobutyric acid-NH40H solvent the two materials have approximately the same mobility.

Reversal of Formation of MurNAc(-pentapeptide Amide)-P-P- lipid to Form UDP-MurNAc-pentapeptide Amide-It has been shown previously that the formation of the first lipid

TABLE VI

Stimulation of peptidoglycan synthesis by ammonia

Peptidoglycan was synthesized and assayed as described previously (12) with ammonia-free substrates (see “Methods and Materials”). Each reaction mixture contained 6 pl of 0.5 M Tris- HCl buffer, pH 8.5, containing 0.017 M MgClt; 1~1 of 0.5 mM UDP- MurNAc-Ala-Glu-r”C-Lys-Ala-Ala (65,000 cpm) ; 0.5 81 of 5 rnM UDP-GlcNAc; 5 ~1 of 20 mM ATP; 1.0 ~1 of 0.05 M 2-mercaptoethanol; particulate enzyme (prepared by grinding cells with alumina; 150 rg of protein) ; and NH&l (adjusted to pH 8.5 with Tris base) as indicated below in a total volume of 30~1. The mixtures were incubated for 1 hour at 25”. After paper chromatography in isobutyric acid-l M NHaOH (5:3), the origins, which contained the peptidoglycan, were cut out and counted in a liquid scintillation spectrometer.

Reaction mixture Concentration of N&Cl in reaction mixture Peptidoglycan

?nM CM 1 0 1,350 2 2.7 3,580 3 6.9 4,520 4 11.8 4,270


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intermediate, MurNAc(-pentapeptide)-P-P-lipid, could be reversed by addition of UMP. Morevoer, if substituents such as glycine were subsequently added to this lipid intermediate, its formation could also be reversed by UMP to form a novel uridine nucleotide (14). Thus, glycine could be added either to MurNAc(-pentapeptide)-P-P-lipid or to GlcNAc-MurNAc- (-pentapeptide)-P-P-lipid. S imilarly, ammonia could be added to either of the lipid intermediates. If a particulate enzyme was incubated for 30 min in the absence of UDP-GlcNAc and then UMP was added for an additional 30 min of incubation, the lipid intermediate was discharged (Table VII). A new nucleotide was formed, with a chromatographic mobility in the isobutyric acid-NHdOH solvent just faster than that of UDP- MurNAc-pentapeptide. The formation of this new compound was dependent on the presence of ammonia. Its formation was virtually eliminated if UMP was present from the beginning of the incubation rather than being added only after 30 min. Under these latter conditions, little lipid intermediate was synthesized either, and hence this observation provides evidence that ammonia cannot be added directly to UDP-MurNAc- pentapeptide.

The following observations identify the new compound as UDP-acetylmuramyl-pentapeptide amide. Its electrophoretic mobility was slower than that of UDP-MurNAc-pentapeptide (Fig. 2). On treatment with snake venom phosphodiesterase, a new compound with the mobility expected for phospho- MurNAc-pentapeptide amide was formed. On treatment with E. coli phosphomonoesterase, no hydrolysis of the nucleotide occurred, but treatment with both phosphodiesterase and phosphomonoesterase yielded a compound, MurNAc-pentapep-

TABLE VII

Conditions for formation of UDP-MurNAc-pentapeptide amide

Each reaction mixture contained the following, in a total volume of 75 ~1: 1 ~1 of 0.5 mM UDP-MurNAc-I%-pentapeptide (40,000 cpm) ; 9 ~1 of 20 mM ATP; 0.5 ~1 of 0.05 M a-mercaptoethanol; 10 pl of 0.5 M Tris-HCl buffer, pH 8.5, containing 0.017 M MgC12; 3.0 pl of RNase (0.2 mg per ml); and 40 ~1 of particulate enzyme (lysostaphin enzyme, 8.5 mg of protein per ml). Other conditions are indicated below. The mixtures were incubated for 1 hour at room temperature, heated for 1 min to lOO”, spotted on Whatman No. 3MM filter paper, and chromatographed in isobutyric acid-l M ammonia (5:3) as described previously. The spots of UDP-MurNAc-pentapeptide amide (RF 0.22; RF of UDP- MurNAc-pentapeptide 0.17) and lipid intermediate (RF 0.9) were located by radioautography, cut out, and counted in a liquid scintillation spectrometer.

Radioactivity incorporated into

Conditions UDP-MurNAc-

pentapeptide amide Lipid intermediate

cPm

1. 0.5 M NH&l, 5 pl; no UMP 2. 0.5 M NH&l, 5 pl; 50 mM UMP,

0.3 ~1, added at zero time.. 3. 0.5 M NH&l, 5 ~1; 50 mM UMP,

0.3 ~1, added after 30 min of incubation

4. No NH&l; 50 mM UMP, 0.3 b.1, added after 30 min of incubation

850

510

2,750

490 I

CPm 9,220

190

620

280

a b c d e j

FIG. 2. Degradation of UDP-MurNAc-Ala-Glu(amide)-rJC- Lys-Ala-Ala by snake venom phosphodiesterase and by E. coli phosphomonoesterase and HCl. The reaction mixtures contained the following: (a) UDP-MurNAc-Ala-Glu(amide)-i%-Lys-Ala- Ala (11,000 cpm; prepared in a large scale experiment similar to that of Table VII, tube 3, and further purified by electrophoresis) and 11.3 ~1 of 0.5 M Tris-HCl buffer, pH 8.5, in a total volume of 34 ~1; (b) same as a plus 1~1 of snake venom phosphodiesterase (5 mg per ml); (c) same as a plus 1 pl of E. coli alkaline phosphomonoesterase; (d) same as a plus 1~1 of snake venom phosphodiesterase and 1 pl of E. coli alkaline phosphomonoesterase; (e) K-UDP- MurNAc-Ala-Glu(amide)-i4C-Lys-Ala-Ala (11,000 cpm) plus HCl (final concentration, 0.1 N) in a total volume of 52 ~1; (f) UDP-MurNAc-Ala-Glu-i4C-Lys-Ala-Ala plus HCl (final concentration, 0.1 N) in a total volume of 52~1. Mixtures a to d were incubated for 2 hours at 37”; e and f, for 45 min at 60”. Mixtures e and f were neutralized after incubation by addition of 5.1 ~1 of 1.0 M KHCO, solution. All mixtures were spotted on Whatman No. 3MM filter paper, and the components were separated by electrophoresis as described under “Assay of Amidation Reac- tion.” A radioautogram of the electrophoretic separation is shown.

TABLE VIII

Utilization of UDP-MurNAc-pentapeptide and UDP-MurNAc- pentapeptide amide for synthesis of lipid intermediate

Each reaction mixture contained the following, in a total volume of 72 ~1: 12 ~1 of 0.5 M Tris-HCl, pH 8.5, containing 0.017 M MgClt; 0.5 pl of 2-mercaptoethanol; 1 pl of 5 mM UDP-GlcNAc; 9 ~1 of 20 mM ATP; 40~1 of particulate enzyme (340 pg of protein) ; NH&l as indicated; and the uridine nucleotide indicated below. The mixtures were incubated for 1 hour at 25” and were theu extracted with butanol-6 M pyridinium acetate, pH 4.2 (2: 1) (see “Assay of Amidating Enzyme”). The extracts were washed twice with 60 pl of water, combined, and counted on filter paper in a liquid scintillation spectrometer.

Nucleotide NHKl, 0.2 M Radioactivity in crude lipid extract

rl CM 1. UDP-MurNAc-pentapeptide

(25,000 cpm). 5 20,120 2. UDP-MurNAc-pentapeptide

amide (26,000 cpm). 0 98

tide amide, with the same electrophoretic mobility as disaccharide-pentapeptide amide.

When UDP-MurNAc-pentapeptide amide was used as a substrate in the forward reaction in the presence of UDP- GlcNAc, the observed velocity of formation of the lipid intermediates was less than 1% of that which occurred when UDP-


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TABLE IX Inhibition of amidation reaction bv deoxycholate and octanol

The assay was performed as described in the text.

Sane

Potassium deoxycholate

Octanol

‘oncentration

%

0.036 0.071 0.107 0.220

0.41 0.86

Disaccharide- pentapeptide

Disacchari+

amide formed pe;Frmr$de

cp” cpnt 30,823 18,009

20,845 25,422 1,701 25,697 1,046 25,294

599 21,793

2,093 5,031 2,402 6,542

0 25 50 75 IW 125 no 175

FdDl Numtel

FIG. 3. Separation of the amidated and nonamidated forms of disaccharide(-pentapeptide)-P-P-lipid by partition chromatography on Sephadex G-25. The solvent system used consisted of 100 volumes of water, 100 volumes of ethanol, 100 volumes of benzene, and 30 volumes of I-butanol. For details see “Methods and Materials.” A : 0, separation of a mixture of miinly amidated lipid intermediate (Fractions 25 through 40) containing some amide-free lipid (Fractions 50 through 65), prepared in a normal incubation mixture; l , lipid intermediate (amide-free) obtained from an ammonia-free reaction mixture after 5 min of incubation. B, separation of the amide-free lipid intermediate (Fractions 55 through 75) obtained by blocking the amidation reaction with deoxycholate. The nature of the material eluted only by sub- sequent application of methanol in B is not known, but it may represent the same material eluted earlier, which had been trailing off the column.

MurNAc-pentapeptide was the substrate (Table VIII), This corresponds to the observation that UDP-MurNAc-pentapeptide-pentaglycine, the nucleotide obtained by reversal of the reaction after glycine addition, was also a poor substrate in the forward reaction (14).

it is clear that amidation can occur prior to the incorporation of

Inhibitors of Amidation Reaction-The antibiotics which inhibit cell wall peptidoglycan synthesis had no effect on the amidation reaction. The following were examined: ristocetin (40 pg per ml), vancomycin (40 pg per ml), bacitracin (57 pg per ml), and penicillin G (66 M per ml). RNase (20 pg per ml), which completely inhibits incorporation of glycine into the lipid intermediates (14), also had no effect on the amidation. Thus, . _

glycine into the lipid intermediate. Deoxycholate, at a concentration of O.l%, completely blocked the amidation reaction. The concentration for 50% inhibition was about 0.05% (Table IX). At these low concentrations deoxycholate had no effect on the synthesis of disaccharide(-pentapeptide)-P-P-lipid. Octanol blocked both the synthesis of the lipid and its amidation.

Preparation of Amidated and Nonamidated Forms of Disaccha- ride(-pentapeptide-P-P-lipid-In order to examine directly the amidation of the lipid intermediate, it was necessary to prepare nonamidated intermediates. Preliminary experiments indicated that the two lipids could be partially separated by careful countercurrent distribution with 10 test tubes and butanol-water as the solvent and aqueous phases. As a consequence, partition chromatography on a column of Sephadex G-25 was attempted. The Sephadex was first loaded with the aqueous phase, and the organic phase of the solvent system was used as the mobile phase (see “Methods and Materials”). With the solvent mixture water-ethanol-benzene-1-butanol (100: 100:100:30), the two forms of the lipid were clearly eluted at different positions, the amidated form being eluted before the nonamidated form of the lipid (Fig. 3A, 0). However, when appreciable amounts of both lipids were present, the two peaks overlapped. A complete separation was obtained by doubling the bed volume of the column (to 40 ml) and by reducing the amount of 1-butanol in the solvent mixture to 25 volumes, which resulted in slower elution (Fig. 4B).

Two methods were available for synthesizing the nonamidated lipid: (a) carrying out the incubation mixture in the absence of ammonia and (b) adding deoxycholate to inhibit the amidation step. Both procedures have been used. When the reaction was carried out in the absence of ammonia, the percentage of the amidated form could be reduced to low levels only with very short incubation times, with consequently low yields of the

5WO- WOO- A Moo- 2CQO- 1000 -

L 0 b 0

I 1 0 P5 50 75 100 125 150 175

FIG. 4. Separation of the amidated and nonamidated forms of disaccharide(-pentapeptide)-P-P-lipid by partition chromatog-

__..

raphy on Sephadex G-25. The solvent system used consisted of 100 volumes of distilled water, 100 volumes of ethanol, 100 volumes of benzene, and 25 volumes of 1-butanol (see “Methods and Materials”). A, elution of the same amide-free lipid (made with deoxycholate) as shown in Fig. 3B with this more slowly eluting solvent system. For B, a column was used which was twice as long as that in Figs. 3, A and B, and 4A. The lipid mixture separated was obtained from an ammonia-free reaction mixture (no deoxycholate added) after 50 min of incubation. The first peak (Fractions 50 through 65) represents the amidated lipid; the second Desk (Fractions 90 through llO), the amide-free lipid.


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Issue of February 25, 1968 G. Siewert and J. L. Strominger 789

nonamidated lipid (Fig. 3A, 0). With longer incubation times, the yield of the nonamidated lipid was much greater, but it contained 10 to 25% of the amidated lipid, owing to failure to remove trace amounts of ammonia from the incubation mixture. These two forms could, however, be separated by partition chromatography (Fig. 4B; compare the 20% of amidated lipid in this figure with the greater than 95% in Fig. 34, 0).

When the nonamidated form of the lipid was synthesized by blocking the amidation step with deoxycholate, the lipid intermediate obtained was eluted from the partition column with the system containing 30 volumes of l-butanol at about Fraction 65 (Fig. 3B), just after the amide-free lipid obt,ained in the absence of ammonia (Fig. 3A). When the solvent system containing 25 volumes of l-butanol was used, which elutes the lipids more slowly, the amide-free lipid prepared in the presence of deoxycholate was not eluted at all by this solvent and was eluted only by application of methanol to the column (Fig. 4A). It is not known how these forms of the lipid intermediate differ

TABLE X Amidation of isolated, amide-free lipid-P-P-disaccharide-X’-

pentapeptide

Lipid I was prepared by using ammonia-free substrates and separating the lipid mixture by partition chromatography. Lipid II was obtained by blocking the amid&ion reaction with deoxycholate. The reaction mixtures were similar to those described in the text, but they contained amide-free lipid-P-P-disaccharide- r*C-pentapeptide (17,000 cpm) instead of the two uridine nucleotide substrates and ATP was added as indicated. The assay of amidation was performed as described.

Lipid

I i-l CM

Amide-free I - 664 + - 6,832

Amide-free II

i 4000

&ooo

dl .z 2 2000

8 ::

e 1000

Disaccharide- pentapeptide

CPm

9,382 5,903

10,221

10,147 6,106 8,488

‘, a 0 I5 30 45 60 75 0 2 4 6 8 IO 12 14 16

minutes of incubdiin pi of portkulole sruyme

FIG. 5. Proportionality of the amidation reaction with isolated lipid intermediate to time and amount of enzyme. The composition of the reaction mixtures and the conditions of the assay were the same as those given in Table XI, tube 1, except for the different incubation times and enzyme concentrations given in the figure.

TABLE XI

Requirements for amidatiow of isolated, amide-free disaccharide (-pentapeptide) -P-P-lipid

A solution of disaccharide(-W-pentapeptide)-P-P-lipid (labeled in both n-alanyl residues) in ethanol (15 ~1; 920 cpm per ccl), which had been prepared by blocking the amidation with deoxycholate (see “Methods and Materials”), was evaporated in each tube before addition of the other constituents of the reaction mixture. The complete reaction mixtures contained the following, in a total volume of 50 ~1: 10 ~1 of 0.5 M Tris-HCI buffer, pH 8.5; 0.5 ~1 of 0.5 M MgClz; 0.5~1 of 0.5 M KCl; 1.0 ~1 of 0.05 M 2-mercaptoethanol; 10.0 b1 of 0.02 M ATP; 3.0~1 of 0.5 M NH&I (adjusted to pH 8.5 with Tris base); 1.0 pl of RNase (2 mg per ml); and particulate enzyme (31 mg of protein per ml; 8.6 pl for tubes 1 through 6; 6.0~1 for tubes 7 through 10). The particulate enzyme had been prepared by lysing cells with lysostaphin in the presence of lo+ M MgClz and had been washed withMg++-free buffer. The reaction mixtures were incubated for 1 hour at 25” and were then extracted three times with 1-butanol-6 M pyridinium acetate, pH 4.2 (2:l) (75 J,50 ~1, and 50 ~1). The extracts of each tube were combined (without washing) and were assayed for content of disaccharide- pentapeptide amide as described under “Results.”

Disaccharide- Omissions from complete reaction mixture pentapeptide amide

1. None...................... 2. Enzyme. . . 3. ATP.. 4. NH&l.. 5. KCl. . 6. Mercaptoethanol.

CM CPS 4,740 5,090

560 10,950 475 9,670

1,640 8,220 4,450 5,410 4,490 5,340

7. None. 3,040 8. MgClz. . 1,130 9. MgCle; 5 rnM CaCh added.. 3,260

10. MgCh; 6 mM MnCh added.. 3,410

-

_ Disaccharide- pentapeptide

5 6 7 8 9 PH

6,790 8,220 7,720 7,440

FIG. 6. pH dependence of the amidation reaction of the isolated lipid intermediate. The reaction mixtures and conditions of the assay were the same as those given in Table XI, tube 1, except for the different buffers. For pH 5 to 7, 0.1 M Tris acid-maleate buffer was used; for pH 7 to 9, 0.1 M Tris-NC1 was the buffer.

from each other, but one obvious possibility is that the amide-

free lipid prepared in the presence of deoxycholate is a form to which deoxycholate remains bound during the procedures used for its preparation.


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$90 Biosynthesis of the Peptidoglycan of Bacterial Cell Walls. XI Vol. 243, No. 4

;lmidution of Isolated Amide-free Lipid Intermediate-Both the amide-free disaccharide(-pentapeptide)-P-P-lipid prepared in the absence of ammonia and that prepared by addition of deoxycholate to the incubation mixture served as acceptors of ammonia when they were used to replace the uridine nucleotide substrates in the reaction mixture. In both cases, the amidation reaction required ATP and was inhibited by a low concentration of deoxycholate (Table X). The rate of the reaction was nearly the same as that seen when the uridine nucleotide substrates were used to generate the lipid acceptor.

The amidation of the isolated lipid was proportional to time and amount of enzyme (Fig. 5). The reaction was absolutely dependent on ATP, and virtually completely dependent on NH&l. It was stimulated about 3-fold by addition of MgC&, as well as by MnClt or CaClt (Table XI). The pH optimum was about 8 (Fig. 6).

DISCUSSION

These experiments then show that the amidation of the (Y- carboxyl group of glutamic acid in the peptidoglycan of bacterial cell walls occurs by an ATPdependent amidation reaction which utilizes free ammonia and a lipid intermediate, either GlcNAc- MurNAc(-pentapeptide)-P-P-lipid or MurNAc(-pentapeptide)- P-P-lipid, as substrates. The reaction is analogous to the addition of glycine to the Lu-carboxyl group of glutamic acid with particulate enzyme from Micrococcus Zysodeikticus (15). Both reactions are inhibited by deoxycholate at a similarly low concentration, which does not inhibit the formation of the lipid intermediates from the uridine nucleotide substrates. An attempt was made in the present study to identify the products derived from ATP in the synthesis. However, there was exten- sive utilization of ATP in the absence of the specific substrate, which prevented detection of any specific utilization in the amidation reaction. A similar difficulty was encountered in the study of the glycine-adding enzyme (15). In that case, indirect evidence was obtained that ADP and inorganic phosphate are the products derived from ATP.

The total amount of lipid synthesized was great,ly stimulated by the presence of ATP at a concentrat,ion much below that required to stimulate the amidation reaction (see Table II). This stimulation of the over-all reaction by ATP had been observed previously (12), but from the present experiments it is clear that the stimulation must be due to something in addition to the utilization of ATP in the amidation step. Possibly there is some reaction in the synthesis of lipid intermediates in which ATP participates as an allosteric activator, or possibly ATP is required to keep the lipid in phosphorylated form so that it can

act as an acceptor of P-MurNAc-pentapeptide from the uridine nucleotide substrate.2

After the initial formation of MurNAc(-pentapeptide)-P-P- lipid in S. aura, at least three modifications of this intermediate occur prior to its utilization for peptidoglycan synthesis: (a) addition of GlcNAc, (b) addition of the pentaglycine chain, and (c) the amidation of the cr-carboxyl group of glutamic acid. In vitro these three steps can apparently occur in any sequence, and no data presently available indicate which, if any, may be the preferred or obligatory sequence in tivo.

Acknowledgment--We are very grateful to Dr. Michio Matsu- hashi, who provided much helpful advice during the initial stages of the investigation, especially regarding the development of the assay procedure.

REFERENCES 1. STROMINGER, J. L., ANDERSON, J. S., MATSUHASHI, M.,

DIETRICH, C. P., MEADOW, P., KATZ, W., SIEWERT, G., AND GILBERT, J. M., in E. NEUFELD AND V. GINSBURG (Editors), Methods in enzymology, Vol. VIII, Academic Press, New York, 1966, p. 473.

2. DIETRICH, C. P., MATSUHASHI, M., KATZ, W., SIEWERT, G., THOMAS, P., AND ANDERSON, J. S., Fed. Proc., 26,588 (1966).

3. TIPPER, D. J., AND STROMINGER, J. L., Proc. Nat. Acad. Sci. U. S. A.. 64, 1133 (1965).

4. TIPPER, D.‘J., KATZ, k., AND STROMINGER, J. L., Biochemistry, 6, 921 (1967).

5. PETIT, J.-F., MUNOZ, E., END GHUYSEN, J.-M., Biochemistry, 6, 2764 (1966).

6. ANDERSON, J. S., MATSUHASHI, M., H~SKIN, M. A., AND STROMINGER, J. L., Proc. Nat. Acad. Sci. U. S. A., 63, 881 (1965).

7. ANDERSON, J. S,, MATSUHASHI, M., HASKIN, M. A., AND STROMINGER, J. L., J. Biol. Chem., 342. 3180 (1967).

8. SCHINDLER, C. A., AND SCHUHARDT, V. T., Proc. Nat. Acad. Sci. U. S. A., 61, 414 (1964).

9. BROKER, H. P., ZYGMUNT, W. A., YOUNG, J. R., AND TAVOR- MINA, P. A., Biochem. Biophys. Res. Commun., 19,383 (1965).

10. ITO, E., AND STROMINGER, J. L., J. Biol. Chem., 237, 2689 (1962).

11. ITO, E., AND STROMINGER, J. L., J. Biol. Chem., 23’7, 2696 (1962).

12. ANDERSON, J. S., MEADOW, P. M., HASKIN, M. A., AND STROM- INGER, J. L., Arch. Biochem. Biophys., 116, 487 (1966).

13. ITO, E., AND STROMINGER, J. L., J. Biol. Chem., 239, 210 (1964). 14. MATSUHASHI, M., DIETRICH, C. P., AND STROMINGER, J. L.,

J. Biol. Chem., 242, 3191 (1967). 15. KATZ, W., MATSUHASHI, M., DIETRICH, C. P., AND STROM-

INGER, J. L., J. Biol. Chem., 242, 3207 (1967). 16. HIG~SHI, Y., SIEWERT, G., AND STROMINGER, J. L., Fed. Proc.,

in mess.

* This stimulation is now known to be due to the presence of an ATP-dependent phosphokinase which catalyzes the conversion of free Cjs-isoprenoid alcohol in the particulate enzyme of S. aureus to the lipid carrier, Css-isoprenoid alcohol phosphate (16).


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Gerhard Siewert and Jack L. StromingerSTAPHYLOCOCCUS AUREUS

OF THE ISOGLUTAMINE AMIDE GROUP IN THE CELL WALLS OF Biosynthesis of the Peptidoglycan of Bacterial Cell Walls: XI. FORMATION

1968, 243:783-790.J. Biol. Chem.

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Biosynthesis of the Peptidoglycan of Bacterial Cell Walls · uridine nucleotide substrates in the amidation reaction. Some properties of the reaction are reported. The peptidoglycan