Synthetase enniatin

Similarly, iterative NRPSs operate in a linear fashion but utilize at least one domain or module multiple times for the synthesis of a single NRP product. Thus, peptides assembled by iterative synthetases contain short, repeating units of peptide building blocks. In such systems, the terminal PCP-TE (or infrequendy PCP-C) didomain is responsible for both condensation of the repeating peptide units and chain release from the assembly line. NRPs biosynthesized in this manner include enniatin, enterobactin, bacillibactin, " gramicidin and the depsi-peptides valinomycin and cereulide. Of these examples, condensation of the precursor peptides for both enterobactin and gramicidin S has been extensively studied and will be discussed in detail. 

In a limited number of NRPSs, the final module terminates in a specialized C domain that catalyzes chain release through amide bond formation. Modules of this type are found in the synthetases involved in the biosynthesis of enniatin, vibriobactin, cyclosporin/ HC-toxin/ and PF1032A. Unlike TE termination, this method of chain release does not utilize an acyl-ester intermediate. Most likely, the chain termination precursor is presented to the C domain as an aminoacyl-5-PCP substrate. Most of these specialized C domains... 

Figure 2 Order and organization of enniatin synthetase and cyclosporin synthetase as deduced from gene sequence and biochemical characterization. Symbols in the adenylateforming modules (black boxes) indicate the corresponding activated amino acids. M stands for A -methyltransferase domain. Condensation domains are represented by white boxes. (A) Top Structure of enniatin synthetase. EA represents the D-Hiv-activating module EB represents the L-valine-activating module D-Ehv is D-2-hydroxyisovaleric acid. Bottom Structural features of the wild-type A -methyltransferase domain M of esynl. The black boxes indicate conserved motifs which can be found within methyltransferases and A -methyltransferase domains of peptide synthetases (see also Fig. 3). The numbers indicate the amino acid position in the sequence of Esyn. (B) Structure of cyclosporin synthetase. Abu = L-a-aminobutyric acid Bmt = (4A)-4-[(E)-2-butenyl]-4-methyl-L-threonine.

Figure 6 (A) Scheme of events of dipeptidol condensations on enniatin synthetase and...

Beauvericin is a structural homolog of enniatins in which the branched-chain L-amino acid is substituted by the aromatic amino acid L-phenylalanine. Beauvericin synthetase, which has been isolated from the fungus Beauveria bassiana [54] and various strains of Fusaria [55], strongly resembles Esyn with respect to its molecular size and the reaction mechanism. In contrast to Esyn, which is only able to incorporate aliphatic amino acids, beauvericin synthetase exhibits high substrate specificity for aromatic amino acids such as phenylalanine. This capability is obviously caused by mutational alterations in the adenylation domain of this enzyme. 

As in the case of enniatins, a 350-kDa depsipeptide synthetase, PF1022 synthetase (PFsyn), is responsible for PF1022A synthesis. The enzyme is capable of synthesizing all known natural cyclooctadepsipeptides of the PF1022 type (A,... 

C Hacker, M Glinski, T Hombogen, A Doller, R Zocher. Mutational analysis of the N-methyltransferase domain of the multifunctional enzyme enniatin synthetase. J Biol Chem 275 30826-30832, 2000. 

M Herrmann, A Haese, R Zocher. Effect of disruption of the enniatin synthetase gene on the virulence of Fusarium avenaceum. Mol Plant-Microbe Interact 9 226-232, 1996. 

R Zocher, U Keller, H Kleinkauf. Enniatin synthetase a novel type of multifunc-... 

A Billich, R Zocher. Constitutive expression of enniatin synthetase during fermentative growth of Fusarium scirpi. Appl Environ Microbiol 54 2504-2509, 1988. 

A Haese, M Schubert, M Hermann, R Zocher. Molecular characterization of the enniatin synthetase gene encoding a multifunctional enzyme catalysing A-methyl-depsipeptide formation in Fusarium scirpi. Mol Microbiol 7 905-914, 1993. 

A Haese, R Pieper, T von Ostrowski, RZocher. Bacterial expression of catalytically active fragments of the multifunctional enzyme enniatin synthetase. J Mol Biol 247 116-122, 1994. 

A Billich, R Zocher, H Kleinkauf, DG Braun, D Lavanchy, HK Hochkeppel. Monoclonal antibodies to the multi enzyme enniatin synthetase. Biol Chem Hoppe-Seyler 368 521-529, 1987. 

R Pieper, A Haese, W Schroder, R Zocher. Arrangement of catalytic sites in the multifunctional enzyme enniatin synthetase. Eur J Biochem 230 119-126, 1995. 

RPieper, HKleinkauf, RZocher. Enniatin synthetases from different Fusaria exhibiting distinct amino acid specificities. J Antibiot 45 1273-1277, 1992. 

A Doller, A Haese, R Zocher. Molecular cloning of the amino acid activation domain of enniatin synthetase from Fusarium sambucinum. Symposium Enzymology of Biosynthesis of Natural Products, Abstract 66, Technische Universitat Berlin, September 22-25, 1996. 

Although most modules of peptide synthetases show a strict specificity towards natural amino acids, some modules can incorporate alternative amino acids into their products. For example, the enniatin synthetase shows a relatively broad substrate specificity for hydrophobic amino acids (L-Val, L-Leu, L-Ile). Both nonmethylated and methylated versions can be processed to generate de-psipeptides in a cell-free system [202]. Likewise, cyclosporins with a variety of alternative amino acids have also been synthesized from cell-free cyclosporin synthetase preparations [203]. Various enniatins and cyclosporins can also be isolated from fermentation broths [204,205]. 

Bahbar CJ, Vaillancourt FH, Walsh CT. Generation of D amino acid residues in assembly of arthrofactin by dual condensa-tion/epimerization domains. Chem. Biol. 2005 12 1189-1200. BilUch A, Zocher R. N-Methyltransferase function of the multifunctional enzyme enniatin synthetases. Biochemistry 1987 26 8417-8423. 

Some nonribosomally synthesized peptides recognize and incorporate carboxy acids other than amino acids into nonribosomally synthesized peptides (Marahiel et al., 1997 von Dohren et al., 1997). Examples of non-amino acid substrates include 2-hydroxyvaleric acid in AM-toxin and enniatin, 2,3-dihydroxybenzoic acid in enterobactin, and lysergic acid in the ergopeptines. The incorporation of non-amino acids into peptides appears to be catalyzed by typical peptide synthetase modules (Haese et al., 1993 Gehring et al., 1998 Johnson et al., 2000). 

In table 1 the present state of characterization of several enzyme systems discussed in this publication is shown. While synthetases for gramicidin S, alamethicin or enniatin are available in purified or partially purified forms, the biosynthesis of actinomycins and valinomycin cannot yet be studied in vitro presumably,because the preparation of a cell free extract of these membrane bound systems needs more subtle and advanced techniques of membrane isolation and fragmentation. A promising approach for the investigation of such systems has been found in the study of protoplasts from the producer organism of these antibiotics. 

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