Understanding the early stages of peptide formation during the biosynthesis of teicoplanin and related glycopeptide antibiotics

Abstract

The biosynthesis of the glycopeptide antibiotics (GPAs) demonstrates the exceptional ability of nonribosomal peptide (NRP) synthesis to generate diverse and complex structures from an expanded array of amino acid precursors. Whilst the heptapeptide cores of GPAs share a conserved C terminus, including the aromatic residues involved cross-linking and that are essential for the antibiotic activity of GPAs, most structural diversity is found within the N terminus of the peptide. Furthermore, the origin of the (D)-stereochemistry of residue 1 of all GPAs is currently unclear, despite its importance for antibiotic activity. Given these important features, we have now reconstituted modules (M) 1-4 of the NRP synthetase (NRPS) assembly lines that synthesise the clinically relevant type IV GPA teicoplanin and the related compound A40926. Our results show that important roles in amino acid modification during the NRPS-mediated biosynthesis of GPAs can be ascribed to the actions of condensation domains present within these modules, including the incorporation of (D)-amino acids at position 1 of the peptide. Our results also indicate that hybrid NRPS assembly lines can be generated in a facile manner by mixing NRPS proteins from different systems and that uncoupling of peptide formation due to different rates of activity seen for NRPS modules can be controlled by varying the ratio of NRPS modules. Taken together, this indicates that NRPS assembly lines function as dynamic peptide assembly lines and not static megaenzyme complexes, which has significant implications for biosynthetic redesign of these important biosynthetic systems.

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

“…37,38 The utility of this approach is not limited by the requirement to maintain E-domain activity, as C-domains are not always exclusively active on the correct peptide stereochemistry as seen previously for teicoplanin M3 and M7, for example. 14,25 Having seen the importance of E-domains within modular exchange strategies, we also tested domain exchange experiments investigating the linkers with the E-domains found in the M4 and M5 modules from the Tcp11 protein. Here, we demonstrated that the construct possessing the C-E domain inter-modular linker (IML) connecting M4 with M5 retains activity but not one with the IML connecting M5 with M6.…”

“…Whilst in the context of the M5-6 + M3a + M4 pathway this could possibly be explained by other as yet unidentied intermodule interactions, in the experiments where synthetic tripeptide was loaded on M3 and incubated with M5 the evidence for N-terminal extension appears unambiguous. Whilst unexpected, it should be noted that there is a general lack of structural information concerning the presentation of acceptor substrates within Cdomains, 5,41,[43][44][45] and that coupled with the reported exibility of the NRPS assembly line (even within fused modules), 25,41,42,46 there is no evidence that the attack of an acceptor peptide onto a donor amino acid is explicitly prevented. This intriguing result highlights the importance of obtaining further structural snapshots of the NRPS C-domain in relevant catalytic states, and is further underlined by the diverse range of catalytic activities performed by domains derived from Cdomains.…”

“…This is consistent with previous results that have showed the M3 C-domain is able to extend incorrectly congured peptides with little effect on extension rate. 25 Engineering modular interactions across fused E-and Cdomains Next, we wanted to further explore how to hybridise modules across the interface between E-and C-domains within multimodular proteins. We concentrated on the M4/M5 interface, as signicant differences in peptide hydrolysis in M4 (high) vs. M5 (low) had previously been published.…”

“…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.…”

Functional Cross-Talk of MbtH-Like Proteins During Thaxtomin Biosynthesis in the Potato Common Scab Pathogen Streptomyces scabiei

“…Several studies have demonstrated a role for these proteins as chaperones in the NRPS assembly line. In these reports, the soluble production of one or more NRPS A-domains in Escherichia coli was shown to be reduced or abolished in the absence of the MLP that is from the same biosynthetic pathway as the NRPS, suggesting that the MLP (called the cognate MLP) is required for the proper folding of the A-domain protein ( Imker et al, 2010 ; Boll et al, 2011 ; McMahon et al, 2012 ; Zolova and Garneau-Tsodikova, 2012 , 2014 ; Kaniusaite et al, 2020 ). Additionally, some MLPs have been shown to influence amino acid activation by the corresponding NRPS.…”