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

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.

Influence of condensation domains on activity and specificity of adenylation domains

“…With this in mind, we reviewed recombinant NRPSs created in our lab to identify functional artificial BGCs showing an unexpected behaviour, like C domains accepting noncognate substrates or altered A domain activation profiles not matching the profiles observed in the natural context (Bozhüyük et al, 2018;Bozhüyük et al, 2019a;Bozhüyük et al, 2021). Indeed, the examples identified do not support the idea that C domains generally have strict selectivity but can at least accept a range of substrates with similar physicochemical properties -supporting insights obtained from the latest solved crystal structure data (Izoré et al, 2021). Nevertheless, especially in the presence of promiscuous A domains, we occasionally observed changes in the substrate activation profiles or the preference of an alternative substrate over the WT substrate observed in situ.…”

“…In sum, these results are indicative for the importance of a functional C-A didomain interface for the activity and specificity of A domains. The gathered in vitro data of both assays along with insights from recent literature data (Baunach et al, 2021;Booth et al, 2021;Calcott et al, 2020;Izoré et al, 2021;Stanišić et al, 2021) provide evidence for an extended gatekeeping function for the C domains upstream of A domains rather than strict intrinsic selectivity. Similar results have also been reported previously for mono-and multi-specific modules that either strictly incorporate leucine or arginine or incorporate chemically diverse amino acids in parallel into microcystin (Meyer et al, 2016).…”

“…1c) (Keating et al, 2002). Together, both subdomains are forming two opposite tunnels that lead from the donor-T and acceptor-T domain binding sites to the conserved key catalytic-residues containing active site motif HHxxxDG (De Crécy-Lagard et al, 1995;Izoré et al, 2021;Keating et al, 2002;Süssmuth and Mainz, 2017). Very early on, biochemical characterizations showed that C domains exhibit a strong stereochemical selectivity for the donor-T domain bound substrate ( L CL, D CL) and a significant side-chain selectivity for the acceptor-T domain bound substrate ( L CL, D CL) (Belshaw et al, 1999;Linne and Marahiel, 2000).…”

“…Very early on, biochemical characterizations showed that C domains exhibit a strong stereochemical selectivity for the donor-T domain bound substrate ( L CL, D CL) and a significant side-chain selectivity for the acceptor-T domain bound substrate ( L CL, D CL) (Belshaw et al, 1999;Linne and Marahiel, 2000). Nevertheless, the exact role and especially how C domains contribute to determining NRPS specificity is still unclear and subject to debate (Baunach et al, 2021;Calcott et al, 2020;Izoré et al, 2021).…”

“…(IV) recent structural data found that C domains do not have a distinct pocket to select the acceptor-T domain bound side chain during peptide assembly, but that residues within the active site motif may instead serve to tune substrate selectivity (Izoré et al, 2021).…”