Evolutionary and functional analysis of an NRPS condensation domain integrates β-lactam, D-amino acid, and dehydroamino acid synthesis

Nonribosomal peptide synthetases (NRPSs) are large, multidomain biosynthetic enzymes involved in the assembly-line-like synthesis of numerous peptide natural products. Among these are clinically useful antibiotics including three classes of beta-lactams: the penicillins/cephalosporins, the monobactams, and the monocyclic nocardicins, as well as the vancomycin family of glycopeptides and the depsipeptide daptomycin. During NRPS synthesis, peptide bond formation is catalyzed by condensation (C) domains, which couple the nascent peptide with the next programmed amino acid of the sequence. A growing number of additional functions are linked to the activity of C domains. In the biosynthesis of the nocardicins, a specialized C domain prepares the embedded beta-lactam ring from a serine residue. Here, we examine the evolutionary descent of this unique beta-lactam-synthesizing C domain. Guided by its ancestry, we predict and demonstrate in vitro that this C domain alternatively performs peptide bond formation when a single stereochemical change is introduced into its peptide starting material. Remarkably, the function of the downstream thioesterase (TE) domain also changes. Natively, the TE directs C terminus epimerization prior to hydrolysis when the beta-lactam is made but catalyzes immediate release of the alternative peptide. In addition, we investigate the roles of C-domain histidine residues in light of clade-specific sequence motifs, refining earlier mechanistic proposals of both beta-lactam formation and canonical peptide synthesis. Finally, expanded phylogenetic analysis reveals unifying connections between beta-lactam synthesis and allied C domains associated with the appearance of D-amino acid and dehydroamino acid residues in other NRPS-derived natural products.

Automated summary

Nonribosomal peptide synthetases (NRPSs) are large, multidomain enzymes involved in the assembly-line-like synthesis of peptide natural products, including clinically useful antibiotics. The condensation (C) domains in NRPSs catalyze peptide bond formation during synthesis. Research has shown that in certain cases, the function of C domains can be altered, providing insights into beta-lactam synthesis and canonical peptide synthesis mechanisms.