Tyrocidine structure

Figure 10 N M R structural analysis of carrier domains. Three conformations of the PCP domain from tyrocidine synthetase (brown box) and the NMR structure of the related AGP domain from a polyketide synthase. The star symbol signifies the position of the conserved phosphopantetheinylated serine residue. The protein ribbon representations are rainbow colored from red (N-terminus) to violet. PDB codes A/H state, 2GDW H-state, 2GDX A-state, 2GDY AGP, 2AF8.

Our approach has been essentially empirical in nature with less emphasis on the theoretical. We have isolated single substances, proved their purity, and determined their covalent structure by classical methods of organic chemistry we have then used these substances of molecular weight ranging from 1,000 to 14,000 as model solutes for the study of conformation and intermolecular interaction. Solutes of special interest have been gramicidin SA (2), bacitracin A (3), polymyxin B, and the tyrocidines A, B, and C (4). All are cyclic antibiotic polypeptides. The first three behave in aqueous solution as reasonably ideal solutes and do not associate, but the tyrocidines associate strongly and are interesting models for the study of association phenomena. Other model solutes of... 

R Dieckmann, M Pavela-Vrancic, E Pfeifer, El von Dohren, El Kleinkauf. The ade-nylation domain of tyrocidine synthetase 1 structural and functional role of the interdomain linker region and the (S/T)GT(T/S)GXPKG core sequence. Eur J Biochem 247 1074-1082, 1997. 

Figure 2 A selection of nonribosomal peptides. Chemical and structural features that contribute to the vast diversity of this class of metabolites are highlighted Heterocycle (bacitracin), lactone (surfactin, daptomycin), ornithine and lactam (Tyrocidine), sugar, chlorinated aromats, C-C crosslink (Vancomycin), N-formyl groups (Coelichelin and linear gramicidin), fatty acid (daptomycin), dihydroxybenzoate and trimeric organization (bacillibactin), dimeric organization (gramicidin S), and ethanolamine (linear gramicidin).

The antibiotics from B. brevis are of much higher molecular weight and presumably are of more complicated structure. Tyrothrycin was the original solid active material from the culture but it was soon shown to be a mixture. Gramicidin was crystallized from the mixture by the appropriate solvents. From the mother liquor tyrocidine was crystallized. 

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. 

The paradigm for both modular PKSs and NRPSs is that each module incorporates one building block into the natural product (Figures 1 and 2). Within each module there are different domains and each domain catalyzes a specific reaction in the assembly of the metabolic product. Model examples of this colinearity between module and domain organization and metabolite structure for type I modular PKS and NRPS systems are respectively represented by erythromycin and tyrocidine biosyntheses. This colinearity is an important feature that underpins our ability to predict structural features of the metabolic products of novel modular PKS and NRPS systems discovered by genomics. 

Tyrocidine is an NRP antibiotic that is isolated from Bacillus hrevis This natural product is able to permeate the lipid phase of the membrane and perturb the lipid bilayer of a Gram-positive inner cell membrane. Although four tyrocidine analogues have been identified, they are assembled by the same biosynthetic machinery that is able to incorporate different amino acids of structural similarity at specified sites. The tyrocidine biosynthetic system consists of three tyrocidine (Tyc) NRPSs, Tyc A-C (Figure 6). In total, 10... 

The molecular weight of tyrocidine A was found to be about 1,270 . Total hydrolysis and quantitative amino acid analysis revealed the exact composition . Partial hydrolysis, fractionation of the resulting peptide mixture by countercurrent distribution, ion-exchange chromatography and paper-chromatography, followed by sequential analysis, led Paladini and Craig to the structure of tyrocidine A see Table 1.6). 

The structure of the crystalline tyrocidine B was elucidated shortly afterwards in a series of analogous experiments by King and Craig . The only... 

Only recently have structures been proposed for tyrocidine C and for the newly isolated tyrocidine D. 

The isolated TE domain from the tyrocidine (tyc) NRPS has recently been shown to catalyze the macrocyclization of unnatural substrates to generate a variety of cyclic peptides. In conjunction with standard solid-phase peptide synthesis, Walsh and coworkers demonstrated a broad substrate tolerance for peptidyl-N-acetylcysteamine thioesters by the tyrocidine TE [41,42], Cyclization of peptide analogs, where individual amino acids were replaced with ethylene glycol units, was observed with high efficiency. In addition, hydroxyacid starter units were readily cyclized by the isolated TE domain to form nonribosomal peptide-derived macrolactones. More recently, Walsh and coworkers have demonstrated effective cyclization of PEGA resin-bound peptide/polyketide hybrids by the tyrocidine TE domain [43], Utilization of a pantetheine mimic for covalent attachment of small molecules to the resin, serves as an appropriate recognition domain for the enzyme. As peptide macrocyclizations remain challenging in the absence of enzymatic assistance, this approach promises facile construction of previously unattainable structures. 

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... 

Tyrocidine A, CMHMNl30. Separation from the tyrocidine mixture Battersby, Craig, J. Am. Chem. Soc. 74, 4019, 4023 (1952), Structure Paladine, Craig, ibid. 76, 688... 

Tyrocidine B, C H N O,]. Purification and amino acid sequence determination King, Craig, J. Am. Chem. Soc. 77, 6624, 6627 (1955). Synthesis Kuromizu, Izumiya, Experi-entia 26, 587 (1970). Possesses the same structure as tyrocidine A except that L-tryptophan replaces the l phenylalanine. Crystals from methanol + isopropyl ether. 

Tyrocidine C, C H N O,. Separation from the tyrocidine mixture Ruttenberg et al. Biochemistry 4, 11 (1965). Possesses same structure as tyrocidine B except that D-tryp -tophan replaces T>-phenylalanine attached to I.-asparagine. Synthesis Kuromizu, Izumiya. Tetrahedron Letters 1970,... 

In some other peptides, the cyclic structure is formed by the peptide bonds themselves. Two cyclic decapeptides (peptides containing 10 amino acid residues) produced by the bacterium Bacillus brevis are interesting examples. Both of these peptides, gramicidin S and tyrocidine A, are antibiotics, and both contain D-amino acids as well as the more usual L-amino acids (Figure 3.14). In addition, both contain the amino acid ornithine (Orn), which does not occur in proteins, but which does play a role as a metabolic intermediate in several common pathways (Section 23.6). 

Tyrocidins homodetic, homomeiic, cyclic peptide antibiotics active against Gram-positive bacteria. Like the Gramicidins (see), X are produced by Bacillus brevis. Hie structure of tyrocidin A, confirmed by total synthesis, is cyclo-(-Val-Om-Leu-D-Phe-Pro-Phe-D-Phe-Asn-Gln-IJr-). In tyrocidin B, Phe is replaced by Trp. In tyrocidin C, Phe is replaced by Tip, D-Phc7 by D-Hp, and Asng by Asp. Tyrocidin E differs from tyrocidin A by replacement of Asn by Asp, and Tyrio by Phe. 

FIGURE 4,16 Isolated NRPS TEs can catalyze die macrocyclization of NRPs such as tyrocidine A. The TycC TE tolerates structural changes at the positions of tyrocidine A diat are shaded gray, (a) Conventional NRP macrocyclization following NRPS-mediated elongation, (b) Chemical synthesis of the 10-mer acyclic peptide followed by activation as the thioester SNAC for macrocyclization by the isolated TycC TE. (c) Installation of the linker on PEGA resin, followed by solid-phase peptide synthesis and deprotection to yield the immobilized acyclic NRP. The TycC TE accepts the resin-bound peptide as a substrate for macrocyclization. 

To increase the ability of broad-spectrum peptide antibiotics to differentiate bacteria from mammalian cells, a focused library of tyrocidine analogs was synthesized based on known structure-activity relationship information (Figure 11.33). Significant side-chain changes of most constituent residues of the tyrocidine template result in the loss of antibiotic potency or the... 

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