Understanding condensation domain selectivity in non-ribosomal peptide biosynthesis: structural characterization of the acceptor bound state

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 the first 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 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.

An Engineered Nonribosomal Peptide Synthetase Shows Opposite Amino Acid Loading and Condensation Specificity

“…[30][31][32][33] The recently solved crystal structure of a C-domain in complex with the T-domain bound at the acceptor site reveals that a binding pocket which would accommodate the amino acid side-chain is absent. 34 This is in agreement with the outcomes of pyoverdine cluster engineering, which yielded functional chimeras after nonsynonymous Adomain substitutions. 30 Therefore, the importance of C-domain specificity for NRPS engineering is a crucial issue and still under debate.…”