Nonribosomal peptide synthetases (NRPSs) are biosynthetic enzymes that synthesize natural product therapeutics using a modular synthetic logic, whereby each module adds one aminoacyl substrate to the nascent peptide. We have determined five x-ray crystal structures of large constructs of the NRPS linear gramicidin synthetase, including a structure of a full core dimodule in conformations organized for the condensation reaction and intermodular peptidyl substrate delivery. The structures reveal differences in the relative positions of adjacent modules, which are not strictly coupled to the catalytic cycle and are consistent with small-angle x-ray scattering data. The structures and covariation analysis of homologs allowed us to create mutants that improve the yield of a peptide from a module-swapped dimodular NRPS.

Nonribosomal peptide synthetases (NRPSs) are biosynthetic enzymes that produce diverse secondary metabolites, including antibiotics and other therapeutics. They are arranged as an assembly line of modules where each module incorporates one aminoacyl monomer into the nascent peptide. Little is known about the interplay between modules within multimodular NRPSs. We determined five x-ray crystal structures from the first two modules of linear gramicidin synthetase, including the full core dimodular structure showing trans-module delivery of the peptide intermediate during the condensation reaction. The structures, along with small-angle x-ray scattering data, show that adjacent modules undergo massive conformational rearrangements and that relative module positions are not governed by the catalytic cycle. Using the structures and covariation analysis, we bioengineered a module-swapped dimodular NRPS with improved catalytic abilities.