Subtilin, amino acids

A large number of a, 3-didehydro-a-amino acids have been identified as constituents of relatively low molecular weight cyclic compounds from microbial sources. However, the presence of a,p-didehydroalanine in bacterial as well as in mammalian histidine ammonia lyase and in phenylalanine ammonia lyase shows that the occurrence of a,p-didehydro-a-amino acids is not limited to small molecules alone 8 These residues are incorporated in natural sequences by posttranslation modification. a,p-Didehydro-a-amino acids have also been postulated to be precursors in the biosynthesis of several heterocyclic metabolites including penicillin and cephalosporin 9 Other well-known compounds containing ,( -di-dehydro-a-amino acids are nisin 10,11 (a food preservative112 ), subtilin (a broad spectrum antibiotic) 13 and some of the metabolites isolated from Streptomyces strains such as gri-seoviridin 14 ... 

Figure 14 Structures of nisins and subtilin. Dha, dehydroalanine Dhb, dehydrobutyrine Ala-S-Ala, lanthionine Abu-S-Ala, /3-methyllanthionine. Filled circles are substituted amino acids from the corresponding residues of nisin A.

The majority of these substances may be considered unique polypeptides which contain unusual amino acids or amino acids joined in some unusual manner. Antibiotics now definitely known to be in this class are the penicillins (2), gramicidin (2), tyrocidine (1), gramicidin-S 3), bacitracin (4), subtilin (-5), polymyxin (6), aerosporin (7) and others. The penicillins are produced by a mold. The others listed here are produced by related bacteria. Streptomycin is a very important antibiotic therapeutically but it does not belong to the polypeptide class, being a complex amino-sugar derivative. 

Reference has been made repeatedly to ,/3-unsaturated amino acids. In nisin (I) (Figure 2) and subtilin (10) (Figure 2), two residues each of dehydroalanine and one residue each of dehydrobutyrine are present. In these molecules three residues each of lysine are also found. With dehydroalanine and lysine present in the same molecules, will it be possible to verify also here the mechanistic concept of the addition of w-amino groups of amino acids across the double bond of ,/ -unsaturated amino acids (Figure 10) and demonstrate the formation of lysinoalanine ... 

When nisin, fragments of nisin (still containing dehydroalanine and lysine), and subtilin were treated under basic conditions, the formation of lysinoalanine was observed in each case. Treatment, for instance, of the carboxyl-terminal fragment of nisin (Figure 11) under these conditions (11) (IN N-ethylmorpholine, pH 10.65, 7 days, room temperature) followed by total hydrolysis and amino acid analysis showed the presence... 

One problem of prime importance is the reliable determination of the number of residues of ,/3-unsaturated amino acids in proteins. Direct amino acid analysis subsequent to total hydrolysis of proteins is not feasible. The ,/3-unsaturated amino acids are subject to degradation with the formation of amide (ammonia) and -keto-acid. The numbers and types of ,/3-unsaturated amino acids in nisin (1) and subtilin (10) and in the fragments of the two peptides were, nevertheless, determined by amino acid analysis, only, however, after the addition of mercaptan across the double bonds of dehydroalanine and dehydrobutyrine (19). Using benzylmercaptan, the addition products are S-benzylcysteine (from dehydroalanine) and /3-methyl-S-benzylcysteine (from dehydrobutyrine). The two thioether amino acids are eluted from ion exchange columns of the amino acid analyzer free from interference by other amino acids... 

Figure 16. Determination of the type and quantity of a,/3-unsaturated amino acids in subtilin—note the elution positions of the addition products of benzylmercaptan to dehydroalanine and dehydrobutyrine S-benzylcysteine ana / -...

The ,/ -unsaturated amino acid dehydroalanine is a necessary precursor for the formation of lysinoalanine. The chemical events covered in the preceding discussion took place in the in vitro environment. Do a S-unsaturated amino acids play a role in any in vivo environment other than that of microorganisms From the latter domain have been isolated the peptides richest in ,/ -unsaturated amino acids, nisin (I) and subtilin (10). No direct evidence is available at this time to document convincingly the possible physiological role of ,/ -unsaturated amino acids in higher organisms. That dehydroalanine is the constituent amino acid of a plant protein has been reported for phenylalanine ammonia lyase from potato tubers (22). There is one case on record for the presence of... 

Gross E, Subtilin and nisin The chemistry and biology of peptides with alpha, beta-unsatu-rated amino acids. Walter R, Meienhofer J, eds. Peptides Chemistry, Structure, and Biology. Ann Arbor. Michigan Ann Arbor Science, 1975 31-42. 

A number of antibiotics contain amino acid residues that differ structurally from those found in proteins. Examples are ,y-diaminobutyric acid in the polymyxins, D- -aminoadipic acid in cephalosporin N and C, / -methyl-lanthionine in subtilin, nisin and cinnamycin, a-phenylsarcosine and L-a,lV-dimethylleucine in etamycin, (-)-)-, e-diamino-w-caproic acid ( 8-lysine) in streptothricini , and L-jS-methylaspartic acid (which can be formed from... 

Carson (109) showed that each of the lysine radicals possessed its -NH2 free and that its a-NH2 was linked in the peptide bond by treating subtilin with nitrous acid or with 2,4-dimtrofluorobenzene. The other free —NH2 groups of subtilin are those of the two sulfur-containing dicarboxylic amino acids. However, for each of these latter residues, only one of their —NH2 groups is free, not both. 

Bacitracin has little or no optical activity (43). It is particularly resistant to acid hydrolysis, i.e., after 18 hours in 6 AT HCl, some peptide still remains (43,79). However, its approximate amino acid analysis (43,143) shows the following for each 100 g. of bacitracin, 11 g. of phenylalanine, 9 g. of leucine, 22 g. of isoleucine, 10 g. of glutamic acid, 17 g. of aspartic acid, 9 g. of lysine, 10 g. of histidine, 14 g. of cystine, and 1.5 g. of ammonia. Perhaps bacitracin also contains ornithine or hydroxylysine. The comparison of the bacitracin composition with that of subtilin is of interest, since both are products of B. svbtilis. 

Gross, E., and H. H. Kiltz The Number and Nature of a,p-Unsaturated Amino Acids in Subtilin. Biochem. Biophys. Res. Comm. 50, 559, 1973. 

Gross, E. Subtilin and Nisin The Chemistry and Biology of Peptides with a,P-Unsaturated Amino Acids. In Peptides Chemistry, Structure and Biology Proceedings of the 4th American Peptide Symp. 1975, p. 31. Ed. by R. Walter and J. Meienhofer. 

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