Tyrocidine properties

Many natural products are constrained by macrocyclic motifs, which are often essenhal for natural products to possess the desired biological properties. In the biosynthesis of macrocyclic NRPs and PKs, linear peptides or PKs are often mac-rocyclized by a TE domain located at the C-terminal of multi-modular synthases. For example, in the biosynthesis of the antibiotic tyrocidine A (Tyc A), a linear enzyme-bound decapephde, which is transferred from the last carrier protein (or thiolahon) domain of the Tyc A synthase, is cyclized by an intramolecular Sn2 reachon between the N-terminal amine nucleophile and the C-terminal ester, which is covalently linked to serine residual in the TE domain prior to macro-cyclization (Scheme 7.9) ([35] and references therein). 

The catalytic mechanisms and molecular recognition properties of peptide synthetases have been studied for several decades [169]. Nonribosomal peptides are assembled on a polyenzyme-protein template, first postulated by Lipmann [170]. The polyenzyme model was refined into the thiotemplate mechanism (Fig. 11) in which the amino acid substrates are covalently bound via thioester linkages to active site sulfhydryls of the enzyme and condensed via a processive mechanism involving a 4 -phosphopantetheine carrier [171-173].The presence of a covalently attached pantetheine cofactor was first established in a cell-free system that catalyzed enzymatic synthesis of the decapeptides gramicidin S and tyrocidine. As in the case of fatty acid synthesis, its role in binding and translocating the intermediate peptides was analyzed [174,175]. 

Some antibiotics that have been derived from peptides were mentioned in Chapter l. The biosynthesis of penicillins was discussed in Chapter 8. Many peptide antibiotics are known. Some find clinical applications but others such as gramicidin S (9.7), tyrocidine A (9.8) and polymyxins (9.9) are too toxic for use in humans. Cyclosporin A (Figure 1.4), however, has immunosuppressive properties and it has been used in transplant surgery for this reason rather than for its antibiotic properties. Peptide antibiotics have some non-standard structural features and these may explain in part their antibiotic properties. First, cyclic peptides are not found in animal cells. Secondly, peptide antibiotics usually contain some unusual amino acids they may have the d configuration, be A-methylated or have other non-standard structural features. Clearly, these features are not compatible with direct ribo-somal synthesis. 

Macrocyclic motifs are usually essential for the unique biological properties of natural products. In most cases, linear NRP and PK scaffolds are cyclized to form macrolactones or macrolactams prior to further post-modification. Macrocyclization is usually carried out by cyclases towards the end of elongation. For example, in the biosynthesis of the antibiotic tyrocidine A, a linear enzyme-bound decapeptide is cyclized via an intramolecular SN2 reaction between the N-terminal amine nucleophile and the C-terminal thioester, which is covalently linked to the synthase [reactions (a) and (b), Scheme 8.3] [22], This cyclase shows great versatility. Not only does it catalyze the formation of macrolactams of ring sizes from 18 to 42 atoms from... 

Gause, Brazhnikova, Lancet 247, 715 (1944). More closely related to tyrocidines, q.v., in biological and chemical properties than to true gramicidins, q.v. Structure Synge, Biochem. J. 39, 363 0945) Consden et al, ibid. 40, xliti... 

Aminoacylation assays have revealed that overexpressed peptide synthetases are only incompletely pusttranslationally modified by pantetheine. Tyrocidine synthetase 1 expressed in E. coJi contains about 1.5% holo-enzyme (80). The reduced cofactitr content leads to decreased activities in aminoacylation and epimerization reactions, which require the cofactor (64,80,84)- Some evidence has been obtained that apo-eniymes may slightly differ from holo--en2ymes with respect to catalytic properties (80). Addition of 4 -phos-phopantetheine is thought to be catalyzed by a holo-enzyme synthase utilizing CoA ... 

The discovery of tyrocidine was made by Hotchkiss and Dubos (286) who, in 1940 isolated a polypeptidelike substance (287) from tyrothricin, an extract of B. brevis cultures. This substance, called tyrocidine by them, is insoluble in acetone and ether. An excellent review of Hotchkiss (284) discusses the work done up to 1944 on the extraction, purification, structure, and biological properties of tyrocidine. The work from 1944 to 1949 is discussed by Synge (575). Actually, tyrocidine, as it was described in the early work, is not a single peptide, but is a family of basic homeomeric peptides, certain of which are cyclic, related one to the other, and characterized by the possession of ornithine. 

Another homeomeric basic cyclic peptide was isolated in 1943 by Gause and Brazhnikova (220) from different strains of B. brevis. This peptide, relatively easily isolated, was called Gramicidin S., and is very close, in properties and composition, to the tyrocidine group. Hence it will be discussed with this family of peptides. 

The action of tyrocidin as a detergent substance affecting the properties of the bacterial cell wall has already been discussed above. 

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