Evolutionary dynamics of natural product biosynthesis in bacteria

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.

Structures of a dimodular nonribosomal peptide synthetase reveal conformational flexibility.

Piecing together nonribosomal peptide synthesis.

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 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 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.

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Structures of a non-ribosomal peptide synthetase condensation domain suggest the basis of substrate selectivity.

Nonribosomal peptide synthetases (NRPSs) are involved in the biosynthesis of numerous peptide and peptide-like natural products that have been exploited in medicine, agriculture, and biotechnology, among other fields. As a consequence, there have been considerable efforts aimed at understanding how NRPSs orchestrate the assembly of these natural products. This review highlights several recent examples that continue to expand upon the fundamental knowledge of NRPS mechanism and includes (1) the discovery of new NRPS substrates and the mechanism by which these sometimes structurally complex substrates are made, (2) the characterization of new NRPS activities and domains that function during the process of peptide assembly, and (3) the various catalytic strategies that are utilized to release the NRPS product. These findings continue to strengthen the predictive power for connecting genes to products, thereby facilitating natural product discovery and development in the Genomics Era.

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Refining and expanding nonribosomal peptide synthetase function and mechanism

Nonribosomal peptide synthetases (NRPSs) increase the chemical diversity of their products by acquiring tailoring domains. Linear gramicidin synthetase starts with a tailoring formylation (F) domain, which likely originated from a sugar formyltransferase (FT) gene. Here, we present studies on an Anoxybacillus kamchatkensis sugar FT representative of the prehorizontal gene transfer FT. Gene cluster analysis reveals that this FT acts on a UDP-sugar in a novel pathway for synthesis of a 7-formamido derivative of CMP-pseudaminic acid. We recapitulate the pathway up to and including the formylation step in vitro, experimentally demonstrating the role of the FT. We also present X-ray crystal structures of the FT alone and with ligands, which unveil contrasts with other structurally characterized sugar FTs and show close structural similarity with the F domain. The structures reveal insights into the adaptations that were needed to co-opt and evolve a sugar FT into a functional and useful NRPS domain.

Structural insight into a novel formyltransferase and evolution to a nonribosomal peptide synthetase tailoring domain.

Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes that synthesize a myriad of diverse molecules. Tailoring domains have been co-opted into NRPSs to introduce further variety into nonribosomal peptide products. Linear gramicidin synthetase contains a unique formylation-tailoring domain in its initiation module (F-A-PCP). The structure of the F-A di-domain has previously been determined in a crystal form which had large solvent channels and no density for the minor Asub subdomain. An attempt was made to take advantage of this packing by removing the Asub subdomain from the construct (F-AΔsub) in order to produce a crystal that could accommodate the PCP domain. In the resulting crystal the original packing network was still present, but a second network with the same packing and almost no contact with the original network took the place of the solvent channels and changed the space group of the crystal.

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T.M. Schmeing

Papers

Structures of a dimodular nonribosomal peptide synthetase reveal conformational flexibility.

Structural insight into a novel formyltransferase and evolution to a nonribosomal peptide synthetase tailoring domain.

Manipulation of an existing crystal form unexpectedly results in interwoven packing networks with pseudo-translational symmetry.