Tyrocidine synthetase

From all data available so far on peptide and depsipeptide synthetases, it af >ears that enzymes of bacterial origin such as gramicidin 5 synthetase, tyrocidine synthetase, or actinomycin synthetase (all reviewed in Ref. 61) have subunit structure, whereas fungal enzymes such as SDZ 214-103 synthetase (62) (see Section VI.C), SDZ 90-215 synthetase (63), and cyclosporin synthetase consist of single polypeptide chains. Cyclospcmn synthetase represents the most complex one. Like type 1 polyketide synthases, these enzymes are designated multifunctional polypeptides the bacterial enzymes, on the other hand, are called "multienzyme complexes, analogous to type 11 polyketide synthases (64). 

Trauger, J.W., Kohli, R.M. and Walsh, C.T. (2001) Cyclization of backbone-substituted peptides catalyzed by the thioesterase domain from the tyrocidine nonribosomal peptide synthetase. Biochemistry, 40 (24), 7092-7098. 

In linear NRPSs a product consisting of amino acids is biosynthesized in an N- to C-terminal manner by the multidomain assembly line with a domain organization of A-PCP-(C-A-PCP) i-TE. The initiation module of a linear NRPS lacks a C domain, while the following modules may include any required additional domains. After formation of the full-length peptide, the product is released from the assembly line by a termination domain. Thus, the number and order of amino acids in the peptide directly coincides with the number and order of synthetase modules. Many NRPs are biosynthesized in this manner, and characterized examples include the penicillin tripeptide precursor -(L-0 -aminoadipyl)-L-cysteinyl-D-valine (ACV, Figure 4 (a)), complestatin, cyclosporin, fengycin, surfactin, and tyrocidine. "... 

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.

Early work summarized in Sec. I.B had shown that ACV is made from L-Aad, L-Cys, and L-Val, that 5-(L-a-Aad)-L-Cys (AC) may be converted into ACV, but L-Cys-D-Val is not converted. Likewise, D-Val is not a substrate for ACV synthetase, contrary to the first-characterized NRPS systems of gramicidin S and tyrocidine [2,3], These observations are in agreement with the scheme, except for the incorporation of AC. This dipeptide was later shown to be activated as an adenylate. 

To demonstrate peptide bond formation from aminoacyl adenylates, Dieck-mann [71] has applied the isolated adenylate domain of tyrocidine synthetase 1 to generate various dipeptides. These were obtained from phenylalanyl adenylate with alanine, leucine, leucineamide, and phenylalanine [reaction (16), with A A = amino acid or amine acceptor],... 

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. 

R Dieckmann. Untersuchungen zu struktur/funktionsbeziehungen von peptidsyn-thetasen am modellsystem tyrocidin synthetase 1. Ph.D. thesis, Technical University of Berlin, 1999. 

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]. 

Trauger JW, KohU RM, Mootz HD, Marahiel MA, Walsh CT. Peptide cycUzation catalysed by the thioesterase domain of tyrocidine synthetase. Nature 2000 407 215-218. 

As described above, the C. purpurea LPS complex is unique among eukaryotes in having its activities divided between two different polypeptides. In its two-polypeptide nature, LPS resembles some prokaryotic peptide synthetases such as tyrocidine synthetase and gramicidin synthetase. However, unlike the prokaryotic peptide synthetases consisting of two polypeptides, the available DNA sequence of cppl (Tudzynski et al., 1999) indicates that its product does not begin with a recognizable condensation domain, as would be typical of the receiving synthetase of a dual-polypeptide system (Marahiel et al., 1997 von Dohren et al., 1997). 

John W. Trauger, Rahul M. Kohll, Henning D.Mootz,Mohamed A. MarahielandChristopherT. Walsh, Peptide cyclization catalysed by the thioester domain of tyrocidine synthetase. Nature, 407 (2000), 215-218. 

Peptide antibiotics are mainly produced by different strains of bacteria and fungi (Ref. 2, 21). They have frequently been reported to appear late in the growth phase (Ref. 21). During studies on tyrocidine biosynthesis in Baoiltus brevis, Lee and Lipmann (l8) found that tyrocidine synthetase was formed at stage 2. 

To this end, researchers prepared TEs, excised from the rest of the NRPS, as isolated enzymes and screened their ability to cyclize substrates outside their native synthetase context. For example, the TE domain from the tyrocidine NRPS (Figure 4.16) has been isolated and shown to retain activity. 5 As has been previously discussed, conventional substrates for TE are activated as the phosphopantetheinyl thio ester of a PCP (Figure 4.16a). Analogous to diketide-feeding experiments with PKSs, Trauger and coworkers demonsttated that the phosphopantetheinyl-PCP could be replaced with a simple SNAC thioester and still be utilized by the TycC TE (Figure 4.16b). Thus, the TycC TE will tolerate changes in the activated linker. 

The NDL synthetase reaction was shown to require ATP, Mg, and MTL and to be heat labile. When ( Clpropylhygric acid replaced [ C]propylprolinc in the reaction mixture, there was no incorporation of the label into lincomycin. The pH and temperature optima were determined to be 8.0 and 30 C, respectively. When the crude extract was diluted below a certain critical concentration, the activity diminished dramatically. Enzyme activity was also lost after passing the preparation through a size-exclusion column, but the activity could be recovered by combining specific fractions (51). These studies supported the hypothesis that the nature of the NDL synthetase activity was that of an enzyme complex of readily dissociable, nonidentical subunits. The activity of one of the subunits was identified by a propylproline-dependent PP -ATP exchange assay based on the method used for the tyrocidine synthetase subunit assay (50,52). The peptide antibiotic synthetases of tyrocidine synthetase, edeine synthetase, and gramicidin S synthetase have previously been reported as complexes of readily dissociable, nonidentical subunits (52-54). 

Lee SG, Lipmann F. Tyrocidine synthetase system. Hash, ]H, ed. Methods in Enzymology. Vol, 43. Antibiotics. New York Academic Press, 1975 585-602. 

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