Many medically relevant complex peptides are naturally produced by enzymes called non-ribosomal peptides synthases (NRPSs).1 Within these multi-modular assembly lines, condensation (C) domains perform the central function of chain assembly, typically by forming a peptide bond from two peptidyl carrier protein (PCP)-bound substrates.2 It is crucial to understand the mechanism in the active site of the C-domains to further develop new structurally diverse antibiotics to fight the growing bacterial resistance crisis by enabling the engineering of the NRPS assembly.3
A common feature in any NRPS biosynthesis is the pantetheine linker between the NRPS machinery and the substrate (also in polyketide and fatty acid synthases). Here, we aim to develop and utilize novel chemical tools around this linker to gain a better structural understanding of the condensation domain. A series of coenzyme A (CoA) probes, where CoAs were precursors loaded onto the carrier proteins that afforded the pantetheine linker, were synthesized and loaded on the domains from the fuscachelin machinery4. Structural snapshots of a C-domain in complex with an aminoacyl-PCP acceptor substrate were obtained (1.9Å).5 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 previously uncharacterized 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.