CHAPTER 5 General discussion
1.4 Non-ribosomal peptide synthetases (NRPSs)
1.4.3 NRPS biosynthetic strategies
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This situation prompted interest in developing new prediction methods supported by other approaches, such as the use of hidden Markov Models (HMM). Khurana et al. (2010) applied HMM to functionally classify the acyl-CoA synthetase superfamily members. The results of this work suggest that the application of HMM to classify this superfamily outperforms the predictions based on a restricted number of active site residues (Khurana et al., 2010).
Furthermore, a novel two-mode factor analysis model based on latent semantic indexing (LSI) has recently been published by Baranasic et al. (2013). This model is able to predict the specific amino acid that is activated by the A domain in contrast to a cluster of similar amino acids.
The authors suggest that a detailed comparison of prediction quality against those of the NRPSpredictor, showed that the LSI model performed slightly better and is thus the most accurate method currently available for prediction of A domain substrate specificities (Baranasic et al., 2013).
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Figure 1.18 Example of the module and domain organization, A-PCP-(C-A-PCP)n-1-TE, observed in a linear or type A NRPS, such as the ACV synthetase, which synthesizes the isopenicillin precursor ACV (Mootz et al., 2002).
In the iterative or type B NRPSs, modules are used more than once in an efficient method of assembling multimeric peptide products. Instead of replicating the elongation modules, the entire NRPS is used repeatedly in an iterative manner to construct peptide chains that consist of recurring, short sequences. The product produced by the first cycle of synthesis is stalled on the C-terminal TE domain, thereby regenerating the NRPS for the assembly of the next product of the same amino acid sequence. Oligomerization of the completed product occurs on the TE domain and is released via hydrolysis or macrocyclization, with the latter being the preferred method. Examples of iterative NRPSs are those that synthesize enterobactin (Figure 1.19), gramicidin, enniatin and bacillibactin (Mootz et al., 2002).
The final biosynthetic strategy is known as the nonlinear or type C NRPS, which can, in most cases, be identified from their primary sequence due to the fact that the identified module and
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domain organization will deviate from the classical C-A-PCP domain organization. They are also characterized by their ability to incorporate small soluble molecules that are not covalently tethered to the NRPS template during synthesis, such as the involvement of amines in vibriobactin biosynthesis, instead of PCP-loaded amino acids (Finking & Marahiel, 2004;
Keating et al., 2002). Due to the fact that amines, such as norspermidine in vibriobactin biosynthesis, lack a carboxyl group necessary for their covalent attachment as a thioester, specialized C domains are employed to incorporate amines. The vibriobactin biosynthetic cluster also encodes an unusual tandem arrangement of Cy domains, in which one is responsible for heterocyclic ring formation, while the other’s function remains unclear (Mootz et al., 2002). Furthermore, unusual internal cyclizations are often associated with deviations from the standard domain organization observed in linear NRPSs, such as in the biosynthesis of bleomycin (Mootz et al., 2002).
Other unusual biosynthetic strategies employed by type C NRPSs include utilizing a single domain to catalyse multiple different reactions, such as the cysteine-specific A domain in the yersiniabactin biosynthetic machinery, which is responsible for the loading of three different PCPs (Suo et al., 2001), as well as the existence of a one-domain NRPS involved in the biosynthesis of the antibiotic novobiocin in Streptomyces spheroides. The enzyme, NovL, shares homology with acyl-adenylate forming enzymes of the same superfamily as A domains and catalyzes formation of the peptide bond between 3-dimethylallyl-4-hydroxybenzoic acid and 3-amino-4,7-dihydroxy-8-methyl coumarin in a reaction that is similar to those catalysed by acyl-CoA ligases. NovL is able to activate the carboxy acid of 3-dimethylallyl-4- hydroxybenzoic acid towards the acyl adenylate, but instead of transferring it onto a PCP domain, the acyl adenylate acts as the electrophile for the condensation with the amino group nucleophile of 3-amino-4,7-dihydroxy-8-methyl coumarin (Mootz et al., 2002). It is also interesting to note that free-standing A domains are known to activate aromatic carboxylic acids and transfer (acylate) them to ArCPs, which are fused to isochorismate lyase, an enzyme involved in the synthesis of 2,3-dihydroxybenzoic acid, which is used as a starter unit in this type of synthetase (Crawford et al., 2011; Schmoock et al., 2005).
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Figure 1.19 Example of the iterative or type B NRPS as observed in the biosynthesis of enterobactin. Three Dhb-Ser-S-ppant intermediates are produced on the two modules of the enterobactin NRPS and oligomerized and cyclized on the TE domain (Mootz et al., 2002).
Additionally, fragments of NRPS assembly lines such as A-PCP didomains or separate, but adjacently encoded A and PCP domains are found in the absence of any other NRPS machinery (Fischbach & Walsh, 2006; Ullrich & Bender, 1994). It was discovered that these A-PCP didomains and freestanding A/PCP domains do in fact activate a specific L-amino acid as an aminoacyl adenylate, which then acts as a substrate for a partner enzyme to chemically modify the β-or γ-carbon of the thioesterified aminoacyl intermediate. Consequently, it has been inferred that the use of didomains or freestanding domains is a strategy to sequester a portion
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of the pool of proteinogenic amino acids in order to modify them into a nonproteinogenic form, which can be used in the biosynthesis of extremely diverse secondary metabolites (Fischbach
& Walsh, 2006).
The nonlinear NRPSs are impressive examples of how microbial producers are able to modify assembly-line organizations and operations in order to evolve novel combinations of the enzymatic components to generate new natural products (Fischbach & Walsh, 2006; Schwarzer et al., 2003).