Synthetase surfactin

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 11 Structurai representatives of the core NRPS domains from X-ray crystallographic analysis, (a) Two conformations (brown box) of A domains differing in the orientation of the subdomains. The top structure (PDB code, 1 AMU) is postuiated to be the conformation responsible for activating the amino acid and the lower (PDB code, 3CW9) for loading the amino acid onto the phosphopantetheine arm. (b) X-ray structure of the VibH condensation domain (PDB code, 1L5A) and (c) the TE domain from surfactin synthetase (PDB code, 1JMK) are also illustrated in ribbon format.

The termination module of surfactin synthetase is a 144 kDa four-domain enzyme responsible for the incorporation of the final amino acid (L-Leu) into the surfactin peptide and subsequent cyclization of the resulting product. The structure of the TE domain of this construct was previously solved. In the recently determined 2.6 A X-ray structure of the C-A-PCP-TE construct, the entire protein chain is evident in the electron density maps. " " The structural folds of the individual domains in this module are similar to structures of monomeric domains (Figure 13). The deviations observed in this multidomain structure include a slight difference in the hinge region of C domain subdomains and an orientation of the subdomains of the A domain that is not consistent with the open or closed conformations previously described. The A domain contains... 

Figure 13 X-ray structure of the four-domain termination moduie of surfactin synthetase (PDB code, 2VSQ). The coioring and representation of the domains is the same as in Figures 11 and 12. A cartoon diagram of the reiative domain structure is iiiustrated at the right of the two views. Ac and An signify the C-terminai and N-terminai subdomains of the A domain.

The replacement of product amino acid residues by positional alteration of domains [102,103], The cysteine- and valine-specific activa-tion-thiolation didomains of P. chrysogenum ACV synthetase have been successfully inserted into the terminal position of surfactin synthetase in Bacillus subtilis [102], In the case of ACV synthetase, e.g., specific domains could be replaced to generate new tripeptides or to improve the efficiency of poorly incorporated amino acid analogues. 

The alteration of product structures by domain repositioning. The terminal thioesterase domain of surfactin synthetase has been repositioned to obtain acyl-tetra- and pentapeptide fragments in vivo of the cycloheptapeptidolactone [104], In the case of ACV synthetase, repositioning of the thioesterase domain would be expected to lead to dipeptides of the AC type. If this specific thioesterase would only release peptides of the D-configuration, alternative thioesterases from other NRPS systems might work. 

The separation of domains to obtain interacting systems with multiple components. This approach directs efforts to enzymatic combinatorial approaches and permits selective improvements on single domains [85], The thioesterase domain of surfactin synthetase has been separately expressed in a thioesterase-deleted surfactin synthetase system and shown to be functional [105], Thus, specific protein-protein inter-... 

PH Weinreb, LE Quadri, CT Walsh, P Zuber. Stoichiometry and specificity of in vitro phosphopantetheinylation and aminoacylation of the valine-activating module of surfactin synthetase. Biochemistry 37 1575-1584, 1998. 

E Guenzi, G Galli, I Grgurina, E Pace, P Ferranti, G Grandi. Coordinate transcription and physical linkage of domains in surfactin synthetase are not essential for proper assembly and activity of the multienzyme complex. J Biol Chem 273 14403-14410, 1998. 

Bacillussubtilis produces the cyclic peptide antibiotic surfactin (82).This is an acyl-pep-tidolactone composed of a p-hydroxy fatty acid side chain and seven amino acid residues, including a leucine at position 7. The biosynthesis of surfactin is catalyzed by a peptide synthetase consisting of three modules. The first and second modules are carrying three domains, the last module just one domain. The last domain catalyzes the activation and... 

Duitman, E.H. et al. (2007) Novel methods for genetic transformation of natural Bacillus subtilis isolates used to study the regulation of the mycosubtilin and surfactin synthetases. Appl. Environ. Microbiol, I i (11),... 

This targeted domain replacement of srf AC was successfully carried out for three bacterial domains of the grs operon activating Phe, Om. and Leu as well as for the Val-and Cys-activating domains of the fungal aevA gene. Surfactin derivatives produced by the chimeric synthetases were extracted from the cultured broth of different recombinant strains, examined for their hemolytic activity, and analyzed by infrared spectra and mass spectrometry. These studies clearly confirmed the identity of five novel Upopeptides that were produced by the selective domain exchange within the srf A operon. The surfactin isomers were found to carry the desired amino acid residue, as was expected from the corresponding domain replacement. 

D Souza C, Nakano MM, Corbell N, Zuber P. Amino-acylation site mutations in amino acid-activating domains of surfactin synthetase Effects on sutfactin production and competence development in Bacillus subtilis. J Bacterial 1993 175 3502-3510. 

Recent examples of these approaches include ACV synthetases (122,123). surfactin synthetases (59,109,124,125), HC-toxin synthetase (126), cyclosporin synthetase (5,47). and enniatin synthetase (103). The difficult part is to provide meaningful amino acid sequence data from the very large proteins, generally to be derived from peptide fragments. 

Vollenbroich D, Kluge B, D Souia C, Zuber P, Vatet J. Analysis of mutant amino acid-activating domain of surfactin synthetase bearing a serine-to-alanine substitution at the site of carboxylthioester formation FEBS Lett 1993 325 220-224... 

Vollenbroich D, Mehta N, Zuber P, Vater J, Kamp RM. Analysis of surfactin synthetase subunits in Srfa mutants of Bacills subtilis Ole B 105. J Bacteriol 1994 176 395-400. 

Calti C, Rodriguez F, Cosinina P, Ptatesi C, Nt iotto R, de Ferra F, Crandi G. Characteti-lation of the surfactin synthetase multienzyme complex. Biochim Biophys Acta 1994 1205 19 28. 

Nonribosomal peptide synthesis means that the peptide is not produced by the tRNA-mRNA mechanism described in Chapter 28, Section 28.6. Each amino acid found in 224 is directly selected for incorporation into the growing peptide chain by one of the domains of surfactin synthetase, shown with the pendant SH groups. Substrate activation occurs after binding the amino acid, and the enzyme catalyzes the formation of an aminoacyl adenylate intermediate using Mg2+-ATP and release of a cofactor. Subsequently, the amino acid-O-AMP oxoester is converted into a thioester by a nucleophilic attack of the free thiol-bound cofactor of an adjacent PCP domain. (Note that ATP is adenosine triphosphate and AMP is adenosine monophosphate see Chapter 28, Section 28.5.)... 

It is noteworthy that nonribosomal peptide synthetase is similarly posttrans-lationally modified by covalent attachment of the 4 -phosphopantetheine group to the peptidyl carrier protein (PCP) [193-198]. While the ACPS can modify various apo-ACPs [167,173,187-189,191,192],it failed to modify PCPs from a variety of peptide synthetases [189]. This led to the discovery of the second family of PPTases [189], such as EntD from E. coli [189, 199, 200], Sfp from Bacillus subtilis [189,200-204], PptT from M. tuberculosis [264], and Gsp from B. brevis [ 189,205,206], required for the biosynthesis of enterobactin, surfactin, mycobactin, and gramicidin S, respectively. In contrast to ACPS, proteins in the latter family, such as Sfp, showed broader substrate specificity, modifying apo-PCPs, apo-ACPs, as well as apo-aryl carrier proteins and utilizing both CoA, acyl CoAs, and CoA analogs [204]. 

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