Eur. J. Biochem. 66, 623 -626 (1976)

Biosynthesis of Gramicidin S with the Aid of Dipeptides by Gramicidin S Synthetase Adolf VON DUNGEN, Joachim VATER, and Horst KLEINKAUF Abteilung Biochemie, Max-Volmer-Institut fur Physikalische Chemie und Molekularbiologie, Technische Universitlt Berlin (Received March 13/ May 4, 1976)

Dipeptides L-phenylalanyl-proline, D-phenylalanyl-proline, prolyl-valine, valyl-lysine, lysylleucine and leucyl-phenylalanine, derived from the sequence of gramicidin S, are substrates of the gramicidin S synthetase. When any of these dipeptides are used to replace the two corresponding amino acids in the reaction assay, cyclodecapeptide antibiotic synthesis occurs, and requires the whole multienzyme system. Active esters, like the thiophenyl and p-nitrophenyl esters of D-phenylalanylproline are unable to promote gramicidin S biosynthesis with the gramicidin S synthetase system or with the heavy enzyme alone. Gramicidin S, the cyclodecapeptide (D-Phe-ProVal-Orn-Leu)z, is synthesized by the multienzyme complex gramicidin S synthetase, by activation of the constituent amino acids as adenylates and subsequent transfer of the aminoacyl adenylates to thiol groups of the enzyme [l - 31. Phenylalanine racemase, the light component of the enzyme complex, activates and racemizes L and D-phenylalanine, and can use both these isomers in the synthesis of the antibiotic. The reaction sequence is initiated when phenylalanine raceinase transfers D-phenylalanine to proline on the heavy enzyme [4]. Then a pantetheine -SH function of the heavy enzyme allows the condensation of the amino acids to take place. In order to achieve further insight into the mechanism involved in biosynthesis of peptide antibiotics, we studied the enzymic synthesis of gramicidin S or related analogs by gramicidin S synthetase with the aid of dipeptides. Two amino acids, following each other in the primary sequence of gramicidin S, were replaced by the corresponding dipeptides (L or DPhe-Pro, Pro-Val, Val-Lys, Lys-Leu, Leu-Phe), thus avoiding one activation and condensation step, which is performed chemically by prior synthesis of the dipeptide. Furthermore we tried to initiate gramicidin S biosynthesis with the heavy enzyme alone, using either D-Phe-Pro, the first intermediate peptide or two of its activated esters. ____ Abbreviations. ONp, p-nitrophenyl ester; SPh, thiophenyl

ester. Enzyme. Phenylalanine racemase, light component of the gramicidin S synthetase (EC 5.3.1.11).

MATERIALS AND METHODS Thin-Layer Chromatography Commercially available silica gel plates, obtained from E. Merck, or poly(ethy1eneimine)-cellulose plates, obtained from C. Schleicher & Schiill, were used for thin-layer chromatography. The following solvent systems were used : (A) butan-1 -ol/acetic acid/water (4/1/4, v/v/v) ; (B) chloroform/methanol/ water (65/25/4, v/v/v) ; (C) ethyl acetate/pyridine/ acetic acidlwater (60/20/6/11, v/v/v/v). Peptides were detected by the ninhydrin colour reaction of free amino groups, by the chlorine/tolidine reagent [ 5 ] , o f under the ultraviolet lamp. Dipep t ides

The dipeptides Pro-Val, Val-Lys . HCl, Lys-Leu . HCl and Leu-Phe and the amino acid derivatives used

in peptide synthesis were pure products obtained from Bachem Inc. (Liestal, Switzerland). The following dipeptides were prepared by the dicyclohexylcarbodiimide method: D-phenylalany1-Lproline hydrobromide was prepared from N-t-butyloxycarbonyl-D-phenylalanine and proline methyl ester hydrochloride ; D-phenylalanyl-L-proline-p-nitrophenyl ester hydrochloride was prepared from N-t-butyloxycarbonyl-D-phenylalanine and proline-p-nitrophenyl ester trifluoroacetate; D-phenylalanyl-L-proline thiophenyl ester trifluoroacetate was prepared by the coupling of N-t-butyloxycarbonyl-D-phenylalanineto proline thiophenyl ester hydrobromide which was synthesized according to Wieland et al. [6].

624

Gramicidin S Synthetase Enzymes were prepared according to Kleinkauf et al. [8] with some modifications [7]. The light and the heavy enzyme were isolated together by DEAEcellulose chromatography, and then separated on a Sepharose 6B column according to their molecular weights.

Biosynthesis of Gramicidin S with the Aid of Dipeptides

able, however, we first established that L-lysine could be substituted for L-ornithine. As shown in Table 1, the lysine analog of gramicidin S was formed in almost the same amount as gramicidin S itself. Thus dipeptides containing L-lysine could be substituted for dipeptides containing L-ornithine. Tables 2 and 3 (experiments 3 - 11) show that cyclodecapeptide antibiotics were formed when two of the five substrate amino acids (L-phenylalanine, Cyclodecapeptide Synthesis L-proline, L-valine, L-ornithine and L-leucine) were by Grarnicidin S Synthetase replaced in the assay mixture by the corresponding dipeptides L-Phe-Pro, D-Phe-Pro, Pro-Val, Val-Lys, For the biosynthesis of peptide antibiotics, 1 pmol Lys-Leu and Leu-Phe, derived from the sequence of ATP, 0.25 pmol EDTA, 25 pmol triethanolamine gramicidin S. The results of the millipore assay were buffer pH 7.8, 5 pmol MgC12, 1 pmol of each of the confirmed by thin-layer chromatography and radioamino acids or dipeptides, and one l4C-labe1led amino scanning. In synthesis experiments 3 - 11 the cycloacid as described in Results, were mixed with the decapeptides formed could be detected by these techwhole multienzyme or with the heavy enzyme alone and niques. incubated at 37 "C for 30 min. For each set of experiThe present experiments show that D-Phe-Pro, the ments described in Results a different enzyme preparafirst peptide detectable in gramicidin S biosynthesis, tion was used. In control experiments one noncan mediate antibiotic synthesis (see Table 3, experiradioactive amino acid was omitted, which ensures ment 11), whereas previous investigations had demonthat no cyclodecapeptide is formed. ~-['~C]Leucine had ~ Ci/mol and L - [ ~ ~ C ] - strated that longer intermediary peptides, like D-Phea specific activity of 2 . 4 lo5 Pro-Val and D-Phe-Pro-Val-Orn could not be incorvaline of 2.8 x lo5 Ci/mol. porated into gramicidin S by multienzyme preparaCyclodecapeptide synthesis was assayed by the tions [lo]. millipore filter method of Gevers et ul. [8]. The milliSince L-Phe-Pro and D-Phe-Pro were both efficient pore assay was corroborated by thin-layer chromatogsubstrates for gramicidin S synthetase (experiments 10 raphy and radioscanning: Ethanol (1 ml) was added and 11 of Table 3), it was of interest to determine to the reaction mixture (170-220 pl) and the solution whether the reactions of the light enzyme could be was evaporated to dryness at 40 "C. This procedure bypassed in the presence of these dipeptides. Tests was repeated and the residue was resuspended in a were therefore performed as described in experiments small amount of HCl/methanol (1% 1 N HCl; v/v). Insoluble protein was removed by centrifugation. The 9 - 11 of Table 3, but with a heavy enzyme preparation clear solution was chromatographed on thin layers of obtained by Sepharose 6 B chromatography, without silica gel, using solvent (A) for development. In selected the light enzyme. Our assay mixtures contained cases, zones of peptide antibiotics were removed from L-Phe-Pro or D-Phe-Pro and the other substrate the thin-layer plates. The cyclodecapeptides were amino acids L-valine, L-ornithine and ~-[l~C]leucine. eluted with methanol and rechromatographed using The radioactivity measured in synthesis experiments solvent systems (B) and (C) for development. Cyclowas not higher than that in the control experiments, and no cyclodecapeptide could be detected by radiodecapeptides synthesized by gramicidin S synthetase scanning or thin-layer chromatography. From these were also identified by measurement of the radioexperiments we conclude that the antibiotic synthesis activity incorporated into the peptide chain due to the with L-Phe-Pro or D-Phe-Pro by the heavy enzyme added radioactive amino acid. Thin-layer plates were scanned in a R. Berthold thin-layer scanner. alone is not possible. Most probably, gramicidin S biosynthesis occurs only after formation of an initiation complex of the heavy enzyme with the racemase. RESULTS AND DISCUSSION The light enzyme exhibits an affinity for the heavy After the formation of the thiol-linked D-phenylenzyme even when not charged with phenylalanine alanyl-proline on the heavy enzyme, the biosynthesis of [ll]. This interpretation is supported by the results of gramicidin S proceeds with the further elongation of Pass et al. [4], who were able to synthesize gramicidin the enzyme-bound polypeptide chains with one-byS in an incubation mixture, which contained the heavy one addition of amino acids. Omission of one amino enzyme loaded with all five substrate amino acids in acid interrupts the sequence and leads to premature the presence of the light enzyme alone. Perhaps the termination [9]. racemase has a complex-forming function with the In the present experiments, dipeptides were subheavy enzyme, together with its ability to transfer stituted for each consecutive pair of amino acids. D-phenylalanine to the heavy component of the Since no ornithine-containing dipeptides were availgramicidin S synthetase.

625

A. von Dungen, J. Vater, and H. Kleinkauf

Table 1, Incorporation of L-lysine into the polypeptide chuin of the cyclodecapeptide antibiotic The reaction mixture contained, in a final volume of 220 pl, 1 pmol of each of the non-labelled amino acids, 835 pmol [14C]leucineand the gramicidin S synthetase complex. Other conditions are described in Materials and Methods. A yield of 100% refers to the total amount of radioactivity of ''C-labelled amino acid in reaction mixture. Yields in Tables 1 3 are estimated from excess of radioactivity over control ~

Expt

Amino acids in reaction mixture

1. Gramicidin S biosynthesis

Phe, Pro, Val, Om, [I4C]Leu

Yield of cyclodecapeptide synthesis

Millipore assay

x

counts/min 10349

2.5 Control

2. Synthesis of bishomo-gramicidin S

Pro, Val, Om, ['4C]Leu

4 369

Phe, Pro, Val, Lys, ['4C]Leu

9 676

Pro, Val, Om. [14C]Leu

4369

2.2 Control

Table 2. Cyclodecapeptide synthesis with dipeptide components of the gramicidin S sequence The reaction mixture contained, in a final volume of 170 pl, gramicidin S synthetase, 1 pmol of each of the non-labelled substrate amino acids or 1 pmol of the dipeptides, and 714 pmol ['4C]leucine(Expts 3 5) or 714 pmol ['4C]valine (Expts 6 - 8). Other conditions are described in Materials and Methods ~

Expt

3. Gramicidin S biosynthesis with [I4C]Leu

Amino acids and peptides in reaction mixture

Phe, Pro, Val, Orn, [I4C]Leu

Millipore assay

Yield of cyclodecapeptide synthesis

counts/min

x

18922 4.7

Control

4. Gramicidin S synthesis with Pro-Val

Pro, Val, Om, [14C]Leu

7 997

Phe, Pro-Val, Orn, [I4C]Leu

17974 ~

Control 5. Bishomo-gramicidin S synthesis with Val-Lys

Phe, Pro-Val, [14C]Leu

3.6

9757

Phe, Pro, Val-Lys, [14C]Leu

15510 2.8

Control 6. Gramicidin S biosynthesis with [14C]Val

Pro, Val-Lys, ['4C]Leu

9000

Phe, Pro, [14C]Val,Orn, Leu

77036

Pro, ['4C]Val, Om, Leu

16616

Phe, Pro, ['4C]Val, Lys-Leu

36 845

Pro, [14C]Val,Lys-Leu

21 823

Pro, ['4C]Val, Orn, Leu-Phe

72 547

['4C]Val, Orn, Leu-Phe

28 397

20.8 Control 7. Bishomo-gramicidin S synthesis with Lys-Leu

5.2 Control 8. Gramicidin S synthesis with Leu-Phe

15.2 Control

An activation of the proline COOH group was achieved by synthesizing the activated esters Dphenylalanylproline thiophenyl ester (D-Phe-Pro-SPh, a thioester) and D-phenylalanylproline p-nitrophenyl ester (D-Phe-Pro-ONp, an oxygen ester). Lipmann and coworkers [12] had shown that activation of

D-phenylalanine as the adenylate and subsequent transfer to an -SH function of the racemase could be substituted by artificially thiosterified analogs of D-phenylalanine. Therefore it might be expected that thiophenyl and nitrophenyl esters of D-Phe-Pro would react with a thiol group of the proline activation site

626

A. von Dungen, J. Vater, and H. Kleinkauf: Biosynthesis of Gramicidin S with the Aid of Dipeptides

Table 3. Initiation of cyclodecapeptide synthesis with L-Phe-Pro or D-Phe-Pro and activated esters The reaction mixture contained gramicidin S synthetase, 1 pmol of each of the non-labelled substrate amino acids or 1 pmol of the dipeptides, and 714 pmol [14C]valinein a final volume of 200 pl. Other conditions are described in Materials and Methods Expt

9. Gramicidin S biosynthesis

Amino acids and peptides in reaction mixture

Millipore assay

Yield of cyclodecapeptide synthesis

counts/min

%

Phe, Pro, [14C]Val, Om, Leu

16266 2.9

~

Control 10. All-L grdmicidin S synthesis with L-Phe-Pro

Pro, [14C]Val,Om, Leu

8 643

L-Phe-Pro, [14C]Val,Om, Leu

18842

L-Phe-Pro, [14C]Val, Leu

11411

2.9 Control 11. Gramicidin S synthesis with D-Phe-Pro

D-Phe-Pro, ['4C]Val, Om, Leu

23 668

D-Phe-Pro, ['4C]Val, Leu

13507

D-Phe-Pro-ONp, ['4C]Val, Om, Leu

12730

D-Phe-Pro-ONp, ['4C]Val, Leu

12147

D-Phe-Pro-SPh, ['4C]Val, Om, Leu

11281

D-Phe-Pro-SPh, ['4C]Val, Leu

10102

3.9 Control 12. Synthesis assay with D-Phe-Pro-ONp Control 13. Synthesis assay with D-Phe-Pro-SPh Control

or with the pantetheine thiol, to form the enzymebound thioester of D-Phe-Pro known to be present in the course of gramicidin S biosynthesis. It also seemed possible that they might react directly with the amino function of valine, which is thioesterified to the heavy enzyme. However, the results of Table 3 (experiments 12, 13) show that all these reactions do not occur, even with the whole gramicidin S synthetase complex, perhaps due to diketopiperazine formation. Thin-layer chromatograms of synthesis experiments 12 and 13 showed an unidentified lipophilic substance, which did not react with ninhydrin. This substance could be a cyclodipeptide, which can be formed easily from dipeptide esters containing proline [13]. Experiments with the active esters and the heavy enzyme alone were also negative. Preliminary studies (Vater, J., v. Dungen, A., unpublished results) show that dipeptides are incorporated intact into the antibiotics. Most probably they are introduced into the reaction cycle at the active center specific for their C-terminal amino acid, overriding the activation site of their N-terminal amino acid. The precise mechanism of these reactions, and the nature of products formed, will be examined in further studies.

We thank Miss B. Kablitz and Mrs K. Struwe for excellent technical assistance. This work was supported by Grant K1 148/11 of the Deutsche ForschunRs~emeinschuft.

REFERENCES 1. Kurahashi, K. (1974) Annu. Rev. Biochem. 43, 445-459. 2. Lipmann, F. (1973) Acc. Chem. Res. 6 , 361-367. 3 . Laland, S. G . & Zimmer, T.-L. (1973) Essays Biochem. 9, 31 - 51. 4. Pass, L., Zimmer, T.-L. & Laland, S. G. (1974) Eur. J. Biochem. 47,607-611. 5. Mazur, R. H., Ellis, B. W. & Cammarata, P. S. (1962) J . Biol. Chem. 237,1619-1621. 6. Wieland, T. & Heinke, B. (1958) Liehigs Ann. Chem. 615, 184-202. 7. Koischwitz, H . & Kleinkauf, H. (1976) Biochim. Biophys. Acta, 429, 1041- 1051. 8. Gevers, W., Kleinkauf, H. & .Lipmann, F. (1968) Proc. Nut1 Acad. Sci. U.S.A. 60, 269-216. 9. Gevers, W., Kleinkauf, H. & Lipmann, F. (1969) Proc. Nut/ Acad. Sci. U.S.A. 63, 1335-1342. 10. Saito, Y. & Otani, S. (1970) Adv. Enzymol. 33, 337-380. 11. Pass, L., Zimmer, T.-L. & Laland, S. (1973) Eur. J . Biochem. 40,43 - 48. 12. Roskoski, R., Ryan, G., Kleinkauf, H., Gevers, W. & Lipmann, F. (1971) Arch. Biochem. Biophys. 143, 485-492. 13. Rydon, H. & Smith, P. (1956) J . Chem. Soc. 3642-3650.

A. von Dungen, J. Vater, and H. Kleinkauf, Abteilung Biochemie, Max-Volmer-Institut fur Physikalische Chemie und Molekularbiologie, Technische Universitat Berlin, FranklinstraRe 29, D-1000 Berlin (West) 10