Issue 1, 2019

The biosynthetic implications of late-stage condensation domain selectivity during glycopeptide antibiotic biosynthesis

Abstract

Non-ribosomal peptide synthesis is a highly important biosynthetic pathway for the formation of many secondary metabolites of medical relevance. Due to the challenges associated with the chemical synthesis of many of the products of these assembly lines, understanding the activity and selectivity of non-ribosomal peptide synthetase (NRPS) machineries is an essential step towards the redesign of such machineries to produce new bioactive peptides. Whilst the selectivity of the adenylation domains responsible for amino acid activation during NRPS synthesis has been widely studied, the selectivity of the essential peptide bond forming domains – known as condensation domains – is not well understood. Here, we present the results of a combination of in vitro and in vivo investigations into the final condensation domain from the NRPS machinery that produces the glycopeptide antibiotics (GPAs). Our results show that this condensation domain is tolerant for a range of peptide substrates and even those with unnatural stereochemistry of the peptide C-terminus, which is in contrast to the widely ascribed role of these domains as a stereochemical gatekeeper during NRPS synthesis. Furthermore, we show that this condensation domain has a significant preference for linear peptide substrates over crosslinked peptides, which indicates that the GPA crosslinking cascade targets the heptapeptide bound to the final module of the NRPS machinery and reinforces the role of the unique GPA X-domain in this process. Finally, we demonstrate that the peptide bond forming activity of this condensation domain is coupled to the rate of amino acid activation performed by the subsequent adenylation domain. This is a significant result with implications for NRPS redesign, as it indicates that the rate of amino acid activation of modified adenylation domains must be maintained to prevent unwanted peptide hydrolysis from the NRPS due to a loss of the productive coupling of amino acid selection and peptide bond formation. Taken together, our results indicate that assessing condensation domain activity is a vital step in not only understanding the biosynthetic logic and timing of NRPS-mediated peptide assembly, but also the rules which redesign efforts must obey in order to successfully produce functional, modified NRPS assembly lines.

Graphical abstract: The biosynthetic implications of late-stage condensation domain selectivity during glycopeptide antibiotic biosynthesis

Supplementary files

Article information

Article type
Edge Article
Submitted
09 Aug 2018
Accepted
10 Oct 2018
First published
10 Oct 2018
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2019,10, 118-133

The biosynthetic implications of late-stage condensation domain selectivity during glycopeptide antibiotic biosynthesis

M. Schoppet, M. Peschke, A. Kirchberg, V. Wiebach, R. D. Süssmuth, E. Stegmann and M. J. Cryle, Chem. Sci., 2019, 10, 118 DOI: 10.1039/C8SC03530J

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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