Isolation of enzyme-bound peptide intermediates in tyrocidine biosynthesis.

The tyrocidines are cyclic decapeptide antibiotics produced by specific strains of Bacillus brevis. Enzyme-bound intermediate peptides, including Phe-Pro-, Phe-Pro-Phe-, Phe-Pro-Phe-Phe-, and others, up to the linear decapeptidyl Phe-Pro-Phe-Phe-Asn-Gln-Phe-Val-Orn-Leu-, can be isolated from appropriately constituted mixtures by precipitation with trichloroacetic acid, and then identified on two-dimensional thin-layer chromatograms after liberation by treatment with alkali. The growing peptide chains are bound to the enzymes by thio ester linkages. The requisite amino acids must be added in the proper order for polypeptide synthesis; T he development of cell-free systems active in tyrocidine formation has furthered our understanding of the mechanism of its biosynthesis. We report, in the preceding paper, that extracts of tyrocidine-producing strains of Bacillus brevis (ATCC 8185), on filtration through Sephadex G-200, yield three complementary fractions which synthesize tyrocidine when supplied with ATP, Mg2+, and the corresponding amino acids (Roskoski et al., 1970). The light component (mol wt 100,000) and the intermediate component (mol wt 230,000) activate phenylalanine and proline, respectively. The heavy component (mol wt 460,000) activates the remaining tyrocidine constituent amino acids. After activation, the aminoacyl residue is transferred from the aminoacyl adenylate-enzyme complex into a thio ester linkage to an enzymic sulfhydryl group, from which it enters into the chain elongation step. The identification of intermediate thio esterlinked peptides in tyrocidine biosynthesis will be the main subject of the present paper. The isolation from the gramicidin S synthesizing system of Phe-Pro-diketopiperazine (Kurahashi, 1961), Phe-Pro-Val (Tomino and Kurahashi, 1964), and Phe-Pro-Val-Orn (Tsuji, 1966; Holm et al., 1966) had indicated that in this process the direction of chain growth appeared to be from phenylalanine to leucine. In gramicidin S biosynthesis we have reported the identification of tri-, tetra-, and pentapeptidy1 hydroxamates from the corresponding peptidylthioenzymes (Gevers et al., 1969; Kleinkauf and Gevers, 1969), and did not find intermediates containing more than five residues, results confirmed by Frgshov et al. (1970). We concluded that cyclization to the decapeptide occurred by headto-tail condensation of two pentapeptidyl thio esters. Simi* From The Rockefeller University, New York, New York 10021. Received August 20, 1970. Supported by Grant GM-13972 from the U. S. Public Health Service. t Recipient of a special fellowship (F03 CA22798) from the U. S. Public Health Service. To whom to address correspondence. if phenylalanine or proline are omitted, no polypeptides are formed. Omission of asparagine stops synthesis at the tetrapeptide stage, even when the succeeding amino acids are present; if ornithine is omitted, synthesis stops at the octapeptide stage. These results indicate that chain growth begins at the D-phenylalanine residue next to proline, and proceeds in order, from amino to carboxyl terminus, until the addition of leucine. Finally, the carboxyl group of this residue, activated as thio ester, reacts with the free amino group of phenylalanine causing cyclization and release of the final product, a relatively slow reaction in the case of tyrocidine. larly, K'urahashi et al. (1969) have reported that Phe-Prodiketopiperazine is formed in extracts which synthesize tyrocidine, suggesting that chain growth begins at the same residue as in the gramicidin S system. Our present data indicate that chain growth in tyrocidine biosynthesis begins at D-phenylalanine and proceeds in order, from amino to carboxyl terminus, to form the linear decapeptide ending with thio ester-linked leucine. This peptide cyclizes relatively slowly to form the final product. The cyclization step in the two decapeptide-synthesizing systems thus appears to differ in a rather interesting way. Experimental Section Enzyme Preparation and Formation of ProteinBound Nascent Peptide Chains. The light, intermediate, and heavy enzyme fractions were prepared from extracts of B. brevis (ATCC 81 85) by ammonium sulfate fractionation and Sephadex G-200 gel filtration as described in the preceding paper (Roskoski et al., 1970). The enzyme-bound peptide chains were separated from reaction mixtures by Sephadex G-50 gel filtration and trichloroacetic acid precipitation, all as described previously (Roskoski et ul., 1970). Radioactivity in the trichloroacetic acid precipitable material was collected on filter disks and determined by the method of Mans and Novelli (1961). Liberation of the Enzyme-Bound Peptide Chains. To break the thio ester link by performic acid oxidation, the method of Frgshov et al. (1970) was used. Alkaline cleavage was carried out as follows : washed precipitates were transferred to tubes and macerated with 50 p1 of water and 2.5 pl of 1.0 N KOH, using a steel spatula. The pH was adjusted to 9-10 if necessary. The tubes were stoppered and placed in a water bath at 90" for 20 min. Then 1 ml of 90% aqueous methanol was added to each tube before centrifugation at 1500g for 10 min. The supernatants were removed with Pasteur pipets and dried in a stream of air at 37". The residues were taken up in 80 4846 B I O C H E M I S T R Y , V O L . 9, N O . 2 5 , 1 9 7 0 P E P T I D E I N T E R M E D I A T E S I N T Y R O C I D I N E B I O S Y N T H E S I S