Structures of a dimodular nonribosomal peptide synthetase reveal conformational flexibility

Abstract

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.

Biochemical characterization of the Nocardia lactamdurans ACV synthetase

“…Despite extensive efforts, including the solution of sub-domain, domain, di-domain and entire modular structures [5,[10][11][12][13][14], the high conformational dynamics and flexibility that characterize NRPS enzymes [15] have rendered structural analysis a considerable challenge. Only recently the first structures of a dimodular NRPS were obtained [16], providing crucial information on the dynamics of inter-domain and inter-module interactions and, ultimately, NRP synthesis.…”

Engineering DNA templated nonribosomal peptide synthesis

“…In crystal structures of the linear gramicidin synthetase A, module dimensions of 85-216 Å have been measured in highly variable conformations. 37 To optimize intermodular spacing, we tested a range of spacers between the ZF recognition sites that would hold the NRPS modules at approximately these distances. Strikingly, with the trimodular system for fPO* formation, we observed a periodic pattern with activity maxima at 10, 20 and 32 bp distance (Figure 4a).…”

“…Comparison with the cyclo-(fP) concentration corrects for the imperfect purity of the covalently linked control protein GrsB123 with a size of 358 kDa ( Figure S6 The spacer length includes the 9 bp ZF binding site and a random sequence of variable length (Table S4) yellow) 36 and crystal structures of an NRPS module (grey) 37 and a ZF (protein in blue, Zn atoms in pink, DNA in grey). 38 (C) In the third module (yellow shade), the ZF is moved to the N-terminus and the DNA spacer is optimized between the C-and N-terminal ZF.…”

What are the principles that govern life?

“…PNPase also plays a pivotal post-transcriptional regulator function in both bacteria and humans. NRPS are complex molecular machines that synthesize small peptides with powerful biological activities [43]. NRPS are widely distributed in bacteria and found sporadically in archaea and eukarya.…”