The crystal structure of the bidomain PCP-C from modules 5 and 6 of the nonribosomal tyrocidine synthetase TycC was determined at 1.8 A resolution. The bidomain structure reveals a V-shaped condensation domain, the canyon-like active site groove of which is associated with the preceding peptidyl carrier protein (PCP) domain at its donor side. The relative arrangement of the PCP and the peptide bond-forming condensation (C) domain places the active sites approximately 50 A apart. Accordingly, this PCP-C structure represents a conformational state prior to peptide transfer from the donor-PCP to the acceptor-PCP domain, implying the existence of additional states of PCP-C domain interaction during catalysis. Additionally, PCP-C exerts a mode of cyclization activity that mimics peptide bond formation catalyzed by C domains. Based on mutational data and pK value analysis of active site residues, it is suggested that nonribosomal peptide bond formation depends on electrostatic interactions rather than on general acid/base catalysis.

Formylation is an important part of ribosomal peptide synthesis of prokaryotes. In nonribosomal peptide synthesis, however, N-formylation is rather unusual and therefore so far unexplored. In this work, the first module of the linear gramicidin nonribosomal peptide synthetase, LgrA1, consisting of a hypothetical formylation domain, an adenylation, and a peptidyl carrier protein domain was tested for formyltransferase activity in vitro. We demonstrate here that the putative formylation domain does indeed transfer the formyl group of formyltetrahydrofolate (fH4F) onto the first amino acid valine using both cofactors N10- and N5-fH4F, respectively. Most important, the necessity of the formylated starter unit formyl-valine for the initiation of the gramicidin biosynthesis was tested by elongation assays with the bimodular system from LgrA. By omitting the formyl group donor, no condensation product of valine with the subsequent building block glycine was detected, whereas the dipeptide formyl-valyl-glycine was found when assayed in the presence of either formyl donor. The proven formylation activity of the first domain of LgrA represents a novel tailoring enzyme in nonribosomal peptide synthesis.

Many microorganisms have evolved an unusual way of producing secondary peptide metabolites. Large multidomain enzymatic machineries, the so‐called nonribosomal peptide synthetases (NRPSs), are responsible for the production of this structurally diverse class of peptides with various functions, such as cytostatic, immunosuppressive, antibacterial, or antitumor properties. These secondary metabolites differ from peptides of ribosomal origin in several ways. Their length is limited to a mere 20 building blocks, roughly, and mostly a circular or branched cyclic connectivity is found. Furthermore, aside from the proteinogenic amino acids, a larger variety of chemical groups is found in these bioactive compounds: D‐configurated amino acids, fatty acids, methylated, oxidized, halogenated, and glycosylated building blocks. These functional and structural features are known to be important for bioactivity, and often natural defense mechanisms are thus evaded. In this article, we describe the enzymatic machineries of NRPSs, the chemical reactions catalyzed by their subunits, and the potential of redesigning or using these machineries to give rise to new nonribosomal peptide antibiotics.

Many microorganisms have evolved an unusual way of producing secondary peptide metabolites. Large multidomain enzymatic machineries, the so-called nonribosomal peptide synthetases (NRPSs), are responsible for the production of this structurally diverse class of peptides with various functions, such as cytostatic, immunosuppressive, antibacterial, or antitumor properties. These secondary metabolites differ from peptides of ribosomal origin in several ways. Their length is limited to a mere 20 building blocks, roughly, and mostly a circular or branched cyclic connectivity is found. Furthermore, aside from the proteinogenic amino acids, a larger variety of chemical groups is found in these bioactive compounds: d-configurated amino acids, fatty acids, methylated, oxidized, halogenated, and glycosylated building blocks. These functional and structural features are known to be important for bioactivity, and often natural defense mechanisms are thus evaded. In this chapter, we describe the enzymatic machineries of NRPSs, the chemical reactions catalyzed by their subunits, and the potential of redesigning or using these machineries to give rise to new nonribosomal peptide antibiotics.Nonribosomal peptide synthetases (NRPSs) compose a unique class of multidomain enzymes capable of producing peptides (1-4). In contrast to the ribosomal machinery where the mRNA template is translated, the order of catalytically active entities within these synthetases intrinsically determines the sequence of building blocks in the peptide product ( Fig. 4.1). As a consequence, generally speaking, each NRPS can only produce one defined peptide product. This is chemically implemented by the fact that all substrates and reaction intermediates are spatially fixed to the synthetase by covalent linkage-thereby eliminating

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