CHAPTER 5 General discussion
1.4 Non-ribosomal peptide synthetases (NRPSs)
1.4.1 Assembly logic of NRP synthesis
1.4.1.5 Editing/tailoring domains
In addition to the essential domains found in every NRPS, further modifications to the final product can be achieved by the action of several editing/modifying domains that act to increase the diversity of NRPs. These so-called modifying domains are not present in every NRPS system, but are nevertheless vital for the proper processing of their designated substrate within their respective synthetase. Inactivation or deletion of these domains usually results in the synthesis of products with severely reduced or no bioactivity.
1.4.1.5.1 Methylation
Certain non-ribosomal peptides such as cyclosporin, enniatin, actinomycin and yersiniabactin possess N-methylated or C-methylated peptide bonds. These modifications are introduced by a methyltransferase (MT) domain, embedded within the canonical fold of an associated A domain, thus making the peptide less susceptible to proteolytic degradation. The MT domain, including both N- and C-methyltransferases, is able to catalyse the transfer of the S-methyl group from S-adenosylmethionine (SAM) to the α-amino group of the thioesterified aminoacyl intermediate in C-A-MT-PCP modules, thereby releasing S-adenosylhomocysteine as a reaction byproduct (Figure 1.14) (Fischbach & Walsh, 2006; Sieber & Marahiel, 2005; Finking
& Marahiel, 2004).
Figure 1.14 The MT domain catalyzes the transfer of the CH3 (Me) group from SAM to the amino group of the aminoacyl-S-intermediate prior to peptide formation with the upstream peptidyl chain (Walsh et al., 2001).
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1.4.1.5.2 Epimerization (control of stereochemistry)
A prevalent structural feature of NRPs is the incorporation of the less common D-amino acids.
Two different strategies are utilized by NRPSs for their incorporation, which may be accomplished simply by an A domain with exclusive specificity towards a D-amino acid or by epimerization of L-amino acids by integrated epimerization (E) domains (Sieber & Marahiel, 2005). The E domains are found on the carboxy terminus of the respective module’s PCP domain and catalyse the racemization of the PCP-bound amino acid or of the C-terminal amino acid of the growing peptidyl chain. Two different types of E domains catalyse these reactions and are known as either aminoacyl epimerases, which can only be part of initiation modules, or peptidyl epimerases, which are part of elongation modules. Due to the fact that E domains are only involved in catalyzing the racemization of their substrates, specific incorporation of the D-amino acid into the growing peptide chain is achieved by the enantiomer-selective donor site of the downstream C domain. The C domain therefore acts as a type of “filter”, which selectively withdraws the correct enantiomer from the pool of L/D-amino acids (Schwarzer et al., 2003). In some rare cases, such as in the arthrofactin synthetase found in Pseudomonas sp.
MIS38, the C domain itself also exhibits epimerization activity in addition to its normal function and it is then known as a “dual C/E” domain (Balibar et al., 2005).
1.4.1.5.3 Reduction domain
A reduction (RE) domain replaces the TE domain in a few NRPS systems, such as in the biosynthesis of gramicidin A in B. brevis and safracin in Pseudomonas fluorescens. RE domains, such as those found in the biosynthesis of myxochelin A in Angiococcus disciformis, catalyse an alternative mechanism of peptide release that involves the NADPH-dependent reduction of the PCP-bound peptidyl thioester to yield an unstable thioacetal intermediate that is converted into a linear aldehyde or alternatively into an alcohol via a second reduction of the thioacetal group (Figure 1.15) (Velasco et al., 2005; Gaitatzis et al., 2001). Another rare type of chain termination strategy is found in some NRPSs, where the RE domain triggers the reductive release, via nucleophilic attack by the free N-terminal amino group, of a highly reactive peptide aldehyde that results in the subsequent formation of a stable macrocyclic imine (Kopp & Marahiel, 2007). RE domains are also known to catalyse the NADPH-dependent
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reduction of thiazoline into thiazolidine by the addition of two electrons, as witnessed in one of the rings in yersiniabactin (Sieber & Marahiel, 2005; Velasco et al., 2005).
Figure 1.15 The RE domain reduces the C-terminal carboxy-group to an aldehyde or to the corresponding alcohol using NADPH as cofactor (Schwarzer et al., 2003).
1.4.1.5.4 Oxidation domain
Oxidation (Ox) domains often appear in the same module as a Cy domain in order to catalyze the oxidation of the hydrolytically labile dihydroheterocyclic thiazolines and oxazolines by two-electron transfer to yield stable heteroaromatic thiazole or oxazole rings (Figure 1.16) (Duerfahrt et al., 2004; Schwarzer et al., 2003). Ox domains can be localized in the accompanying A domain, as is the case in the epothilone synthetase, or they can be C-terminally fused to the PCP domain of the respective module, as witnessed in the bleomycin synthetase (Schwarzer et al., 2003; Du et al., 2000). Furthermore, flavin mononucleotide (FMN) has been verified as a cofactor of Ox domains. FMN is reduced to FMNH2 during the oxidation of the substrate and is subsequently re-oxidised by the reduction of molecular oxygen to superoxide. While the oxidation of thiazoline to thiazole in the epothilone synthetase is catalysed in cis by an FMN-containing Ox domain (a corresponding in cis flavoprotein domain exists in the bleomycin synthetase), a second trans-acting Ox domain has been proposed to be involved in the formation of the second thiazole ring found in the structure of bleomycin A2 (Finking & Marahiel, 2004; Du et al., 2000). The fact that these enzymes are able to act in trans, where they are able to recognise the peptidyl chains on NRPS modules by protein-protein
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interactions, is testament to the enormous structural variation created in these natural products (Walsh et al., 2001).
Figure 1.16 FMN-containing Ox domains catalyze the two electron oxidation of thiazolines and oxazolines to yield stable heteroaromatic thiazole or oxazole rings, respectively (Schwarzer et al., 2003).
1.4.1.5.5 Further modifications
In the case of the linear gramicidin A, the N-terminus of the non-ribosomal peptide possesses a formyl group which is introduced by a formylation (F) domain, localized N-terminal to the A domain of the initiation module. The F domain is responsible for the N-formylation reaction by means of the cofactor N-formyltetrahydrofolate. Other formylated NRPs include coelichelin, whose ornithine residues are believed to be N-formylated in trans and anabaenopeptilide 90-A whose N-terminus exhibits an N-formylated glutamine residue (Lautru et al., 2005; Sieber & Marahiel, 2005).
Surfactin and fengicin are lipopeptides, which represent a subgroup of NRPs whose peptide backbones are N-terminally bound to a fatty acid. An N-terminal C domain, in addition to the A and PCP domain, is usually present in the initiation module of these NRPSs and is thought to catalyse bond formation to the fatty acid.
An aminotransferase (AMT) domain exists within the mycosubtilin NRPS that converts a thioesterified β-ketoacyl intermediate to a covalently tethered β-aminoacyl thioester with
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simultaneous removal of the α-amino group of glutamine to form α-keto-glutamine in a variation of the classic transamination reaction. It was therefore proposed to be responsible for the transfer of an amine group to the β-position of the growing acyl chain. The ability to incorporate amine functionality directly into NRPs creates a functional handle for macrocyclization strategies and acts as a potent hydrogen bond donor, which can significantly alter the biology of a given compound (Fischbach & Walsh, 2006).
All of the modifications that have been introduced thus far are catalysed by domains that are embedded into their respective NRPS modules and almost entirely affect only the peptide backbone. Additionally, certain enzymes such as glycosyl transferases, halogenases and hydroxylases are able to modify the peptide’s side chains and are generally encoded in the same biosynthetic gene cluster.