Mechanisms of activity of Non-Ribosomal Peptide Synthases.

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The search for new potent anti-bacterials as well as the ability to synthesize them in a cost effective way is imperative in view of the world-wide rise of population and of increasing resistance to the prevalent antibiotics. Non-ribosomally synthesized peptides (NRPs) are an important class of natural products that show a wide range of pharmaceutical activity, both as antibacterial and antitumor agents. Their diverse structures form promising scaffolds for the development of new and potent therapeutics. These peptides are synthesized by dedicated molecular machines called Non-ribosomal peptide synthases (NRPS). NRPSs work in an assembly line-like fashion, following the concept of "division of labour". Each NRPS is organized as separate modules, each of which is composed of a set of independently folded domains that catalyse a particular reaction/step in the pathway. The modules are classified as initiation, elongation and termination modules, which respectively start the peptide synthesis, extend the peptide chain and release the final product. The ability of NRPS to synthesize such novel and atypical peptides with varied structural frameworks can be harnessed to our advantage; NRPS modules could be engineered to yield structurally and chemically optimized therapeutics. Clearly, the in silico design of efficient NRPS machinery to synthesize non-natural NRPs requires detailed mechanistic and structural knowledge of the enzyme. The core catalytic domains of the NRPS modules are the adenylation, condensation, and thioesterase domains, which catalyse the substrate activation, peptide bond formation and product release, respectively. The peptidyl carrier (PCP) domain shuttles the growing peptide chain between different reaction centres located either within a module or in separate modules. While the function and selectivity of the adenylation domain has been studied extensively, to date there is no structural information for the condensation domain bound to both the donor and acceptor PCP modules. Consequently, the mechanism and regulation of the condensation reaction remain nebulous. In this project, we will study the simplest natural NRPS, the Tomaymycin synthase. Tomaymycin is synthesized by an initiation module, TomA, and by an elongation/termination module, TomB. We will study the structural basis for the mechanism of the condensation reaction carried out by the C domain of TomB together with the TomA-PCP and TomB-PCP domains loaded with their substrates. To this end, we will use NMR spectroscopy, which is unique in its ability to deal with transient interactions, such as those occurring in the NRPSs.