Structures of a non-ribosomal peptide synthetase condensation domain suggest the basis of substrate selectivity

Thierry Izoré,Joe A Kaczmarski,Colin J Jackson,Manuela Tosin,Athina Gavriilidou,Robert J A Goode,Elizabeth H Krenske,Julien Tailhades,Y T Candace Ho,Ralf B Schittenhelm,Ka Ho Chow,Max J Cryle,Nadine Ziemert,David L Steer,Gregory L Challis

doi:10.1038/s41467-021-22623-0

Abstract

Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.

Highlights

Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products
We demonstrate that C domains do not appear to contain an “A domain-like” side chain selectivity pocket to control their acceptor substrates and resolve how substrates engage with central catalytic residues in C domains, both of which are key unanswered questions central to NRPS-mediated peptide biosynthesis
To elucidate the structure of a C domain with a peptidyl carrier protein (PCP) domain bound in the acceptor site, we screened several systems including a thermophilic example of a PCP2-C3 didomain of the fuscachelin NRPS from the thermophilic organism Thermobifida fusca (Fig. 1a [red rectangle])[30]

Summary

Introduction

Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. The diversity of nonribosomal peptides is due to the combination of an ability to incorporate an expanded range of monomers compared to ribosomal peptide biosynthesis together with extensive modifications of the peptide both during and after chain assembly[2] This is enabled by the modular architecture of NRPSs, which use repeating groups of catalytic domains to install one monomer into the growing peptide (Fig. 1a). The individual domains (and certain didomain complexes that represent metastable points along the catalytic pathway) are less dynamic and can be more readily studied by methods such as X-ray crystallography[7,13,27,28]

Methods

Results

Conclusion