A proof-reading mechanism for non-proteinogenic amino acid incorporation into glycopeptide antibiotics

Abstract

Non-ribosomal peptide biosynthesis produces highly diverse natural products through a complex cascade of enzymatic reactions that together function with high selectivity to produce bioactive peptides. The modification of non-ribosomal peptide synthetase (NRPS)-bound amino acids can introduce significant structural diversity into these peptides and has exciting potential for biosynthetic redesign. However, the control mechanisms ensuring selective modification of specific residues during NRPS biosynthesis have previously been unclear. Here, we have characterised the incorporation of the non-proteinogenic amino acid 3-chloro-b-hydroxytyrosine during glycopeptide antibiotic (GPA) biosynthesis. Our results demonstrate that the modification of this residue by trans-acting enzymes is controlled by the selectivity of the upstream condensation domain responsible for peptide synthesis. A proofreading thioesterase works together with this process to ensure that effective peptide biosynthesis proceeds even when the selectivity of key amino acid activation domains within the NRPS is low. Furthermore, the exchange of condensation domains with altered amino acid specificities allows the modification of such residues within NRPS biosynthesis to be controlled, which will doubtless prove important for reengineering of these assembly lines. Taken together, our results indicate the importance of the complex interplay of NRPS domains and trans-acting enzymes to ensure effective GPA biosynthesis, and in doing so reveals a process that is mechanistically comparable to the hydrolytic proofreading function of tRNA synthetases in ribosomal protein synthesis.Peptide synthesis for tripeptide 3T-CoA, tetrapeptide 4T-CoA and pentapeptide 5T-CoA CoAs was performed manually by 9470 | Chem. Sci., 2019, 10, 9466-9482This journal is

Exploring modular reengineering strategies to redesign the teicoplanin non-ribosomal peptide synthetase

“…The critical factor in these bio-combinatorial experiments is the selectivity of the acceptor site of the upstream C-domain, which in teicoplanin M6 is high due to the need for this domain to gate aminoacyl-PCP modications by trans-acting enzymes. 7,26 In order to generate communication between non-adjacent modules in the teicoplanin NPRS we identied that, in contrast to other NRPS systems, [32][33][34] it is not sufficient to relocate/exchange compatible COM domains at the end of module of interest. Whilst recent strategies that divide fused modules have shown compatibility with COM domain relocation, 35 our results clearly show that larger adjacent domain-domain interaction surfaces are also required to ensure module-module recognition and ensure communication between M5-6 and M3.…”

“…We concentrated on the M4/M5 interface, as signicant differences in peptide hydrolysis in M4 (high) vs. M5 (low) had previously been published. 7 Our hypothesis was that the M4 E-domain was responsible for the signicant hydrolysis seen in in vitro assays, and we wanted to test if Edomain exchange could overcome this. First, we explored the role of the individual M4 and M5 Edomains on peptide hydrolysis.…”

“…Cells were grown and protein expression was induced according to the previously described procedure. 7…”

“…5 C-domains play central roles as gatekeepers within NRPS biosynthesis, where they act to not only control the stereochemistry of the peptide fragment together with the action of neighbouring epimerisation (E)-domainsadditional tailoring domains that supplement the core C-A-PCP domains found within a minimal extension modulebut can also allow modication of the PCP-bound amino acid either directly or via controlling substrate availability for trans-interacting domains such as hydroxylases or halogenases. 6,7 Module expansion by the addition of tailoring domains can extend beyond E-domains, 8,9 with terminal modules typically containing a thioesterase (TE)-domain that then releases the mature peptide product through hydrolysis, dimerisation or cyclisation by attack of an internal nucleophile. 10 Further complex modi-cations are also common within NRPS-biosynthesis, with one important example being the recruitment of multiple external oxygenase enzymes during the cyclisation cascade found in glycopeptide antibiotic (GPA) biosynthesis ( Fig.…”

“…1). 6,7,14,[25][26][27][28] Now, we explore the ability to generate alternate NRPS assembly lines from teicoplanin biosynthesis using a combination of approaches including module hybridisation, re-purposing extension modules as initiation modules and redesigning modules to control intermodule communication through the use of specic domain interaction interfaces. In this way, we present a set of instructions to tackle the reconstitution and reengineering of complex NRPS assembly lines in vitro.…”