Thiotemplate mechanism

H van Liempt, H Palissa, E Pfeifer, T Schwecke, R Weckermann, H von Dohren, H Kleinkauf. ACV-synthetase implications of the amino acid sequence data for the thiotemplate mechanism of peptide biosynthesis. In H Kleinkauf, H von Dohren, eds. 50 Years of Penicillin Application—History and Trends. Prague Publica, 1991, pp 136-144. 

H Kleinkauf, H von Dohren. Thiotemplate mechanism. In TE Creighton, ed. The Encyclopedia of Molecular Biology. New York Wiley, 1990, pp 2539-2541. 

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]. 

I. 1-1) starts with the polymerization of L-a-aminoadipic acid, L-cysteine, and L-valine to the linear tripeptide L-a-aminoadipyl-L-cysteinyl-D-valine(ACV-peptide). This reaction is catalyzed by the ACV-synthase (MW about 420 kD) through the following steps (1) the ATP-dependent activation of these amino acids to bind them as thiolesters, (2) the epimerization of L-valine, and hnally (3) the condensation by a thiotemplate mechanism [99]. 

Thiotemplate mechanism, the central principle of non-ribosomal peptide synthesis. Non-ribosomal peptide synthetases are multienzyme complexes where every single reaction step in the synthesis of the final product is performed by a specialized protein module. The growing peptide chain is connected to the single modules across a thioester bond. The transfer... 

The peptide antibiotic bacitracin is synthesized by the protein thiotemplate mechanism present in Bacillus species... 

The total synthesis of cyclosporin A has been described by R.M. Wenger [38] biosynthetically it is produced by adding appropriate amino acids to the fermentation with the fungus [39] or to cell free preparations therefrom [40]. Various cyclosporins with different activities have been obtained. The activation and connection of the amino acids occurs, as recognized in the synthesis of gramicidin S (p. 208), by the thiotemplate mechanism the methyl groups are introduced into the already formed peptide bonds by S-adenosyl-methionine. 

In 1990, La wen and Zocher (49) described the purification and characterization of the enzyme cyclosporin synthetase, isolated from the mycelium of the strain T. inflatum NRRL 8044 (S 7939/45). This enzyme synthesizes cyclosporin A by a thiotemplate mechanism starting from the precursor amino acids in their unmethylated form, utilizing adenosine triphosphate ATP)/Mg, and with S-adenosyl-L-methionine as methyl donor. The first attempts (50) to establish the cell-firee synthesis of cyclosporin A were not successful, but they led to a partially enriched enzyme that could synthesize the diketopipcr-... 

As already outlined in the first publication on the characterization of cyclosporin synthetase (49), this multifunctional enzyme follows a thiotemplate mechanism. This mechanism, elucidated by the group of Lipmann (65), closely resembles the biosynthesis of fatty acids. In a first step, cyclosporin synthetase activates all constituent amino acids of cyclosporin A in their unmethylated form as aminoacyl adenylates by reaction with ATP (measured by amino acid-dependent ATP-pyrophosphate exchange (49), or directly proved by the use of adenylates as substrates for cyclosporin A biosynthesis (66)) this step also occurs in the ribosomal mechanism. The second step in the nonribosomal mechanism consists of a transesterification of the enzyme-bound activated amino acids onto reactive thiol groups of the enzyme. Thus, cyclosporin synthetase can be radiolabeled by covalent binding of -labeled substrates (e.g., valine and leucine), ATP, and Mg. Fur-... 

In summary, cyclosporin synthetase, the most complex multifunctional enzyme known. so far, catalyzes at least 40 reaction steps 11 aminoadenylation reactions, 11 transthiolation reactions, 7 N-methylation reactions, 10 elongation reactions, and the final cyclization reaction (Figure 7). In a modified thiotemplate mechanism that has been proposed (70), each active site carries its own 4 -phosphopantctheine residue, and therefore, a central 4 -phosphopantetheine arm limited in its geometry is not required (Figure 8). 

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