DEHYDROALANYLLYSINE: IDENTICAL COOH-TERMINAL STRUCTURES IN THE PEPTIDE ANTIBIOTICS NISIN AND SUBTILIN BY ERHARD GROSS, JOHN L. MORELL, AND LYMAN C. CRAIG NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES, NATIONAL INSTITUTES OF HEALTH, AND ROCKEFELLER UNIVERSITY Communicated December 26, 1968 Abstract.-The recent finding of af-unsaturated amino acid residues by Gross and Morrel in the polypeptide antibiotic nisin has stimulated a wider investiga- tion of other antibiotic peptides, particularly those known to contain lanthionine. Subtilin is similar to nisin in that it polymerizes easily and contains lanthionine and ,3-methyl lanthionine. Like nisin it was found to contain a carboxyl terminal dehydroalanyllysine sequence and to be split by the enzyme nisinase. An additional aA-unsaturated amino acid residue was shown to be present in sub- tilin by reaction with excess methyl mercaptoacetate and subsequent hydrolysis and amino acid analysis. Nisin contains three dehydropeptide residues. In spite of countless careful investigations with polypeptides, it is still possible that there are unknown transient and labile linkages that are difficult to detect by the experimental methods presently in use. It is therefore of considerable interest when an unusual and labile linkage is shown experimentally to be present in a naturally occurring polypeptide. The antibiotic polypeptides often seem to have unexpected and labile linkages and are especially interesting in this connection. One such linkage often postulated in peptides but seldom shown definitely to be present is that of an af-unsaturated amino acid residue. This type of residue, namely dehydroalanine, has recently been found to be present in the polypeptide antibiotic nisin.1 2 The chemistry of these substances may well have an interest much beyond the primary objective of establishing their structures. Nisin has been shown to polymerize readily via covalent bond formation,3 and the dehydroalanine residue adds methyl mercaptan4 to give S-methylcysteine. It likewise adds other mercapto derivatives, such as mercaptoacetamide, to give the carbox. amidomethylcysteine residue.' It is of interest that nisin contains lanthionine and /3-methyl lanthionine, since these may be formed easily by the addition of cysteine to dehydroalanine 'or f3-methyldehydroalanine. Indeed, one residue of /3-methyldehydroalanine was found to be present in nisin.3 The dehydroalanine residue was found to be labile to dilute acid. Such treatment of nisin gave a substance identified as pyruvyllysine.' Dehydro- alanyllysine, therefore, is the carboxyl terminal sequence. Even more interest- ing was the finding that while des-(dehydroalanyllysine)-nisin showed no antibiotic activity, such activity was restored when the fragment was incubated with pyruvyllysine under appropriate conditions.' This observation and the polymerization discussed earlier indicate that cleavage of the peptide chain at a,/3-unsaturated amino acids is readily reversible. 952 Downloaded by guest on September 27, 2021 VoL. 62, 1969 BIOCHEMISTRY: GROSS ET AL. 953 The biosynthetic pathways by means of which polypeptide antibiotics are synthesized seem to be a somewhat controversial subject., Apparently a pathway different from that by which polypeptide chains in proteins are syn- thesized is usually followed. The chemistry recently determined', I and out- lined above may be significant in the problem of the biosynthesis of the sulfur- containing polypeptide antibiotics. It also may be significant in suggesting a mechanism for the action of this type of antibiotic. It is now of interest to examine other antibiotic polypeptides in search of dehydropeptide linkages, particularly those containing lanthionines. Cinna- mycin6 and subtilin7 contain these unusual amino acid residues. Subtilin, accordingly, was examined and found to contain dehydropeptide linkages. Other aspects of its chemistry in comparison with the chemistry of nisin are described in this paper. Materials and Methods.-Subtilin was obtained from the Western Regional Laboratory (Dr. Harold Olcott) in 1950. Another sample was received in 1957 (Dr. G. Alderton). It had been purified by silica gel chromatography. The earlier sample was fractionated by countercurrent distribution. Nisin, obtained from Aplin and Barrett Ltd., Yeovil, England, was fractionated by countercurrent distribution. The diffusional sizes of nisin and subtilin were compared by countercurrent thin-film dialysis8 9 in a column 45 cm in length. The membrane was size 20 Visking dialysis casing. For hydrolysis of a-possible dehydropeptide linkage, a sample of subtilin was treated with hydrochloric acid in glacial acetic acid as was previously reported.' The solvent was removed by lyophilization, and an aliquot of the reaction mixture was placed on an ion exchange column of the accelerated system10 of the amino acid analyzer." A sub- stance was eluted from the 0.9 X 60-cm column at the same effluent volume as pyruvyl- lysine' in the amount of 1 Amole/pmole of subtilin. For addition of mercaptan, a sample of subtilin was allowed to react with a 600-fold excess of methyl mercaptoacetate for 13 days at room temperature. The reaction product was separated from the reagent by gel chromatography on Sephadex G-25 and then hydrolyzed with 6 N hydrochloric acid for 24 hr at 108'C. The hydrolysate. gave a band on the amino acid analyzer corresponding to 1.7 residues of S-carboxy- methylcysteine. Discussion.-From the release of pyruvyllysine and the addition of methyl mercaptoacetate with the formation of the methyl ester of S-carboxymethyl- cysteine, we conclude that dehydroalanine is also present in subtilin. Interest- ingly enough, we encounter the same partial structure in both antibiotics, i.e., the carboxyl terminal sequence dehydroalanyllysine. This poses interesting questions with regard to the biosynthesis of these peptide antibiotics and may have significant phylogenetic implications, since the microorganisms from which the antibiotics are isolated are rather different ones. The results of the reaction with methyl mercaptoacetate indicate that subtilin contains two residues of a,$-unsaturated amino acids. This would explain the consistently high amide determinations discussed in the earlier structural work on subtilin."2 The presence of two residues of a,f-unsaturated amino acids is supported by spectral data. The molar extinction coefficient (250 miA, 5 X 10-5 molar solu- tion) of subtilin, less that due to the absorption of aromatic residues, is 7000. By comparison, a fragment of nisin that contains two a,f3-unsaturated amino Downloaded by guest on September 27, 2021 954 BIOCHEMISTRY: GROSS ET AL. PRoc. N. A. S. acids shows a molar extinction coefficient of 6000 at the same wavelengths. 13 The special changes which we earlier observed for subtilin'2 have been rein- vestigated. Nisin does not contain tryptophan; subtilin contains one residue of this amino acid. A mixture of nisin and tryptophan in equimolar amounts showed a UV spectrum similar to that found for subtilin. Moreover, the mixture underwent the same spectral changes as did subtilin when the pH was raised to above 12, namely, a general increase in optical density and broaden- ing of the spectral curve. For both antibiotics, this is due to the formation of dehydroalanine as a result of P,-elimination from lanthionine residues in the alkaline pH range; hydrolysates of samples so treated show a decrease in lanthio- nine and an increase in ammonia. The addition of two residues of dehydro- alanine to the amino acid formula proposed earlierl2 would increase the molecular weight by 138 to approximate a value of 3500. This does not seriously change the good agreement of the amino acid analysis obtained in the earlier work.'2 That nisin and subtilin are very similar in molecular size and shape is clearly shown in Figure 1, which presents results of a comparative study in a new type of thin-film countercurrent dialyzer.9 In this highly efficient dialysis column, which has been improved considerably since the original description, the relative amounts found in the retentate stream and the diffusate stream on a single pass offer a very sensitive comparison- of diffusional size. 1.6 IA- 1.2 705%removal 5.10 . _ \ FIG. 1.-Thin-film counter- current dialysis of subtilin,O--o my), and nisin, -9(250 66.4% removal The left pattern is the reten- tate effluent. The right pattern is the 0.6 nOoX- | - a,~~~~~~~(280diffusate effluent. 0 5 10 15 20 0 5 10 15 20 Tube No. Downloaded by guest on September 27, 2021 VOL. 62, 1969 BIOCHEMISTRY: GROSS ET AL. 955 Subtilin and nisin, both purified by countercurrent distribution, gave straight- line escape plots by the static thin-film dialysis method9 with rates of dialysis corresponding to those expected for compactly folded spherical peptides in the molecular-weight range of 3500. It is doubtful that the subtilin prepara- tion, the molecular weight of which had been determined by the partial sub- stitution method,'4 could have contained appreciable amounts of polymeric forms. However, several years later this same sample, when investigated again by the thin-film dialysis method, gave a result which indicated that considerable polymerization to larger molecular sizes had occurred. This observation and those made during the structural elucidation of nisin3 emphasize the readily occurring polymerization of these peptide antibiotics subsequent to the breakdown of a,4-unsaturated amino acids with the formation of amides and a-keto acids.