CHAPTER 5 General discussion
1.4 Non-ribosomal peptide synthetases (NRPSs)
1.4.1 Assembly logic of NRP synthesis
1.4.1.3 Peptide elongation by the Condensation domain
The C domain is approximately 450 amino acids in length and is responsible for the elongation of the peptidyl chain. C domains are normally localized between each consecutive A-PCP domain and catalyse the condensation reaction between the peptidyl chain tethered to the phosphopantetheinyl arm of the upstream PCP domain and the amino acid bound to the downstream PCP domain (Lautru & Challis, 2004). However, in the first condensation reaction of an NRPS, both these reaction intermediates would typically be aminoacyl groups attached to their respective PCP domains. Furthermore,C domains are absent from modules involved in peptide initiation (Mootz et al., 2002).
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There is little information concerning the exact mechanism of peptide elongation and how the interaction of modules affects the direction of polymerization, but it is thought that peptide bond formation proceeds via the nucleophilic attack of the free α-amino group on the downstream PCP-bound acceptor amino acid on the activated carboxy-thioester of the upstream PCP-bound donor amino acid (Figure 1.9). This reaction facilitates the translocation of the growing peptide chain onto the next module for elongation and structural changes (Bloudoff et al., 2013; Sieber & Marahiel, 2005; Mootz et al., 2002; Marahiel, 1997).
Moreover, this condensation reaction is strictly unidirectional, leading to a downstream- directed synthesis of the NRPS product (Samel et al., 2007).
Figure 1.9 Peptide elongation catalysed by the C domain, which involves an attack of the nucleophilic amine of the acceptor substrate onto the electrophilic thioester of the donor substrate (Sieber &
Marahiel, 2005).
In order to shed light on the catalytic mechanism of the C domain, three structures that contain NRPS C domains have been determined by X-ray crystallography, including a stand-alone C domain (Keating et al., 2002), a C-PCP didomain complex (Samel et al., 2007) and a C-A- PCP-TE termination module (Tanovic et al., 2008). The overall architecture of the C domain revealed a pseudodimeric configuration consisting of both an N-terminal and C-terminal subdomain. The active site is located at the bottom of a “canyon” or “V-shape” formed by the two subdomains and is covered by a “latch” that crosses over from the C to N subdomain
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(Figure 1.10) (Bloudoff et al., 2013; Marahiel, 2009; Finking & Marahiel, 2004). The catalytic centre includes a conserved HHxxxDGxS core motif (where x is any amino acid and defined residues are represented by the single-letter amino acid code), that is also found in dihydrolipoyl transacetylases, chloramphenicol acetyltransferases, and NRPS epimerization and Cy domains (Sieber & Marahiel, 2005; Keating & Walsh, 1999; Marahiel et al., 1997). A model, supported by mutational studies, suggests that the second histidine of this motif, which is located at the bottom of the canyon, may act as a catalytic base promoting the deprotonation of the NH3+ moiety of the thioester-bound nucleophile prior to peptide bond formation.
However, recent pK value analysis of this active site residue suggests that peptide bond formation may depend mainly on electrostatic interactions rather than a general acid/base catalysis (Marahiel, 2009; Samel et al., 2007; Konz & Marahiel, 1999).
Figure 1.10 Structure of the PCP-C didomain from the surfactin synthetase illustrating the active site histidine (His) residue located on the floor of the C-domain “canyon” (Marahiel, 2009).
27 1.4.1.3.1 Heterocyclization domains
The C domain of a module, which catalyzes basic peptide bond formation only, can be replaced by a specialised heterocyclization (Cy) domain that shares striking structural and functional homology to the C domain. The Cy domain combines the condensation function of the C domain with additional heterocyclization and dehydration functions using the side chains of the amino acids cysteine, serine or threonine within the product peptide backbone to produce thiazoline (from cysteine), oxazoline (from serine) or 5-methyloxazoline (from threonine) heterocycles (Walsh et al., 2001).
Cy domains were first identified in 1997 in the cyclic dodecylpeptide antibiotic bacitracin synthetase, produced by Bacillus licheniformis ATCC 10716 (Konz et al., 1997) and were further validated in the biochemical characterisation of yersiniabactin, an iron-chelating virulence factor of Yersinia pestis (Gehring & Walsh, 1998). The catalytic core motif, HHxxxDGxS, of the C domain is modified to DxxxxDxxS in the Cy domain. The conserved aspartate residues are critical for both condensation and heterocyclization (Keating et al., 2002;
Keating & Walsh, 1999). Examples of secondary metabolites produced using Cy domains are epothilone A, myxothiazol and mycobactin A produced by Sorangium cellulosum So ce90, Stigmatella aurantiaca DW4/3-1 and M. tuberculosis, respectively (Figure 1.11) (Duerfahrt et al., 2004).
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Figure 1.11 Heterocyclic ring-containing secondary metabolites from various organisms (the five- membered thiazole and oxazole rings are shaded grey) (Duerfahrt et al., 2004).
The first reaction step catalysed by Cy domains is peptide bond condensation, carried out by the nucleophilic attack of a PCP-bound cysteine, serine or threonine acceptor substrate onto the thioester of the donor substrate. The next step involves the nucleophilic attack by the hydroxyl side chain of serine/threonine or thiol side chain of cysteine onto the carbonyl C atom of the newly-formed peptide bond to yield hemiaminal or thiohemiaminal intermediates and the five-membered heterocyclic ring. The intermediates are subsequently dehydrated to yield the C=N bond and the final oxazoline or thiazoline product (Figure 1.12) (Sieber & Marahiel, 2005; Walsh et al., 2001). In non-ribosomal peptide products such as the glycopeptide antibiotic, bleomycin (produced by ‘Streptomyces verticillatus’ ATCC 15003) or myxothiazol, additional oxidation (Ox) domains convert these heterocycles into more stable oxazole or thiazole rings. Heterocyclic rings are common structural features of NRPs and are important for the interaction with proteins, DNA and RNA, as well for chelating metal ions (Walsh, 2004).
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Figure 1.12 The formation of thiazoline (top of figure) and oxazoline heterocycles (bottom of figure) from cysteine and threonine precursors, respectively (Walsh et al., 2001).