SUMMARY Non-ribosomally synthesized peptides have compelling biological activities ranging from antimicrobial to immunosuppressive and from cytostatic to antitumor. The broad spectrum of applications in modern medicine is reflected in the great structural diversity of these natural products. They contain unique building blocks, such as d-amino acids, fatty acids, sugar moieties, and heterocyclic elements, as well as halogenated, methylated, and formylated residues. In the past decades, significant progress has been made toward the understanding of the biosynthesis of these secondary metabolites by nonribosomal peptide synthetases (NRPSs) and their associated tailoring enzymes. Guided by this knowledge, researchers genetically redesigned the NRPS template to synthesize new peptide products. Moreover, chemoenzymatic strategies were developed to rationally engineer nonribosomal peptides products in order to increase or alter their bioactivities. Specifically, chemical synthesis combined with peptide cyclization mediated by nonribosomal thioesterase domains enabled the synthesis of glycosylated cyclopeptides, inhibitors of integrin receptors, peptide/polyketide hybrids, lipopeptide antibiotics, and streptogramin B antibiotics. In addition to the synthetic potential of these cyclization catalysts, which is the main focus of this review, different enzymes for tailoring of peptide scaffolds as well as the manipulation of carrier proteins with reporter-labeled coenzyme A analogs are discussed.

Daptomycin is a branched cyclic nonribosomally assembled acidic lipopeptide, which is the first clinically approved antibiotic of this class. Here we show that the recombinant cyclization domain of the Streptomyces coelicolor calcium-dependent antibiotic (CDA) nonribosomal peptide synthetase (NRPS) is a versatile tool for the chemoenzymatic generation of daptomycin derivatives. Linear CDA undecapeptide thioesters with single exchanges at six daptomycin-specific residues were successfully cyclized by CDA cyclase. Simultaneous incorporation of all six of these residues into the peptide backbone and elongation of the N-terminus of CDA by two residues yielded a daptomycin derivative that lacked only the beta-methyl group of l-3-methylglutamate. Bioactivity studies with several substrate analogues revealed a significant role of nonproteinogenic constituents for antibacterial potency. In accordance with acidic lipopeptides, the bioactivity of the chemoenzymatic assembled daptomycin analogue is dependent on the concentration of calcium ions. Single deletions of the four acidic residues in the peptide backbone suggest that only two aspartic acid residues are essential for antimicrobial potency. These two residues are strictly conserved among other nonribosomal acidic lipopeptides and the EF-motif of ribosomally assembled calmodulin. Based on these findings CDA cyclase is a versatile catalyst that can be used to generate novel daptomycin derivatives that are otherwise difficult to obtain by chemical modification of the parental tridecapeptide to improve further its therapeutic activity.

Many biologically active natural products have macrocyclic structures. In nonribosomal peptides macrocyclization is commonly achieved via the formation of intramolecular ester or amide bond catalyzed by thioesterase domains during biosynthesis. A unique and so far unknown type of peptide cyclization occurs in the nostocyclopeptide, a macrocyclic imine produced by the terrestrial cyanobacterium Nostoc sp. ATCC53789. In this work we show that a C-terminal reductase domain of the nostocyclopeptide nonribosomal peptide synthetase catalyzes the reductive release of a linear peptide aldehyde and thereby triggers the spontaneous formation of a stable imino head-to-tail linkage. This type of molecular self-assembly induced by the reductive release of reactive aldehydes may be more commonplace in other complex nonribosomal peptides than originally thought.

Here we report the first biochemical characterization of a recombinant nonribosomal peptide cyclase of a streptomycete, the model actinomycete Streptomyces coelicolor A3(2). This bacterium produces the calcium-dependent antibiotic (CDA), which is a branched cyclic macrolactone belonging to the group of acidic lipopeptides. The recombinant CDA3 cyclase from CDA synthetase efficiently catalyzes ring formation of linear peptidyl thioester substrates based on a sequence analogous to natural CDA. Four leaving groups were attached to the C-terminus of the undecapeptide: coenzyme A (CoA), phosphopantetheine, N-acetylcysteamine (SNAC), and thiophenol. The best rates for cyclization were determined for the thiophenol substrate, revealing that chemical reactivity is more important than cofactor recognition. The cyclase catalyzes the formation of two regioisomeric macrolactones, which arise from simultaneous nucleophilic attack of the two adjacent Thr(2) and Ser(1) residues onto the C-terminus of the acyl-enzyme intermediate. This relaxed regioselectivity has not been observed for any other recombinant NRPS or PKS cyclases so far. Substitution of either Ser(1) or Thr(2) by alanine led to selective formation of a decapeptide or undecapeptide lactone ring. In contrast to that, CDA3 cyclase strictly retains stereoselectivity for both nucleophiles, accepting only l-configured Ser(1) and Thr(2) for cyclization. Further, our studies provide evidence for the crucial role of N-terminal fatty acyl groups of lipopeptides in controlling the regio- and chemoselectivity of enzyme-catalyzed macrocyclization. Elongation of the fatty acyl group of our thioester substrate from C(2) to C(6) as in CDA turned the relaxed regioselectivity into a strict regioselectivity, yielding solely the decapeptide lactone ring with a significantly improved cyclization-to-hydrolysis ratio.

The ability to selectively induce a strong immune response against self-proteins, or increase the immunogenicity of specific epitopes in foreign antigens, would have a significant impact on the production of vaccines for cancer, protein-misfolding diseases, and infectious diseases. Here, we show that site-specific incorporation of an immunogenic unnatural amino acid into a protein of interest produces high-titer antibodies that cross-react with WT protein. Specifically, mutation of a single tyrosine residue (Tyr 86 ) of murine tumor necrosis factor-␣ (mTNF-␣) to p-nitrophenylalanine (pNO2Phe) induced a hightiter antibody response in mice, whereas no significant antibody response was observed for a Tyr 86 3 Phe mutant. The antibodies generated against the pNO 2Phe are highly cross-reactive with native mTNF-␣ and protect mice against lipopolysaccharide (LPS)-induced death. This approach may provide a general method for inducing an antibody response to specific epitopes of self-and foreign antigens that lead to a neutralizing immune response.TNF-␣ ͉ unnatural amino acids ͉ vaccination ͉ immunogenicity