Influence of condensation domains on activity and specificity of adenylation domains

Abstract

Many clinically used natural products are produced by non-ribosomal peptide synthetases (NRPSs), which due to their modular nature should be accessible to modification and engineering approaches. While the adenylation domain (A) plays the key role in substrate recognition and activation, the condensation domain (C) which is responsible for substrate linkage and stereochemical filtering recently became the subject of debate - with its attributed role as a "gatekeeper" being called into question. Since we have thoroughly investigated different combinations of C-A didomains in a series of in vitro, in vivo, and in situ experiments suggesting an important role to the C-A interface for the activity and specificity of the downstream A domain and not the C domain as such, we would like to contribute to this discussion. The role of the C-A interface, termed 'extended gatekeeping', due to structural features of the C domains, can also be transferred to other NRPSs by engineering, was finally investigated and characterised in an in silico approach on 30 wild-type and recombinant C-A interfaces. With these data, we not only would like to offer a new perspective on the specificity of C domains, but also to revise our previously established NRPS engineering and construction rules.

Type S Non‐Ribosomal Peptide Synthetases for the Rapid Generation of Tailormade Peptide Libraries**

“…This is of particular interest when it comes to large scale NRPS engineering campaigns, as TE‐domains that are more flexible with respect to peptide length and amino acid sequence have a broader range of application. The approach shown is not only superior to in vitro characterisation in terms of workload, but also much cheaper (no SNAC peptides need to be synthesised), more robust (less spontaneous or autocatalytic side effects), scalable, can be performed in high throughput, and does not suffer from the in vitro bias known for excised NRPS proteins as recently described [32] …”

“…Type S NRPSs can also be used to study C domain specificities and C−A interface compatibilities. Since in the case of GxpS (NRPS‐1 to ‐16), the respective activated and incorporated amino acids are too similar to each other to draw valuable conclusions on C domain specificities ‐ but this was shown previously [17c,32] – we focused on characterising the compatibility of different C−A interface types with each other. In general, the C domains are classified into five different groups based on the reactions they catalyse: L C L , D C L , dualC (C/E), C start , and C term [18] .…”

“…Although we also detected cyclic peptides (22-27) from production cultures of the 8-modular NRPS-12, these cyclic peptides are presumably autocatalytically cyclised shunt products of enzyme-bound hexa-and heptapeptides. NRPS-12 only was capable to hydrolyse the synthesised octapeptides (30)(31)(32)(33)(34), resulting in linear octapeptide derivatives (Figure 1d).…”

“…The approach shown is not only superior to in vitro characterisation in terms of workload, but also much cheaper (no SNAC peptides need to be synthesised), more robust (less spontaneous or autocatalytic side effects), scalable, can be performed in high throughput, and does not suffer from the in vitro bias known for excised NRPS proteins as recently described. [32] Type S NRPSs can also be used to study C domain specificities and CÀ A interface compatibilities. Since in the case of GxpS (NRPS-1 to -16), the respective activated and incorporated amino acids are too similar to each other to draw valuable conclusions on C domain specificities -but this was shown previously [17c,32] -we focused on characterising the compatibility of different CÀ A interface types with each other.…”

Tailoring Enzyme Stringency Masks the Multispecificity of a Lyngbyatoxin (Indolactam Alkaloid) Nonribosomal Peptide Synthetase

“…While the condensation domain (C-domain) may influence substrate activation, [19,22] the role of the C-domain as a gatekeeper has recently been challenged, with C-domains showing relaxed specificity towards substrates with similar physicochemical properties. [28][29][30] Nonetheless, the nature of C-domain substrate tolerance does appear to be variable between pathways, and is particularly important for gating in trans modifications. [31] It is important to note that the activities observed in the assay were substrate activation by both A-domains within LtxA, and in the presence of an MLP due to copurification of LtxB.…”