In silico studies and structure-function mutagenesis have defined general rules for the structural basis of substrate recognition in adenylation domains of NRPSs. These rules can be used to rationally alter the specificity of adenylation domains and to predict from the primary sequence the specificity of biochemically uncharacterized adenylation domains. Such efforts could enhance the structural diversity of peptide antibiotics such as penicillins, cyclosporins and vancomycins by allowing synthesis of 'unnatural' natural products.

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Recently, considerable insight has been gained into the modular organization and catalytic properties of nonribosomal peptide synthetases. However, molecular and biochemical aspects of the condensation of two aminoacyl substrates or a peptidyl and an aminoacyl substrate, leading to the formation of a peptide bond, have remained essentially impenetrable. To investigate this crucial part of nonribosomal peptide synthesis, an in vitro assay for a dipeptide formation was developed. Two recombinant holomodules, GrsA (PheATE), providing D-Phe, and a C-terminally truncated TycB, corresponding to the first, L-Pro-incorporating module (Pro-CAT), were investigated. Upon combination of the two aminoacylated modules, a fast reaction is observed, due to the formation of the linear dipeptide D-Phe-L-Pro-Senzyme on ProCAT, followed by a noncatalyzed release of the dipeptide from the enzyme. The liberated product was identified by TLC, high pressure liquid chromatography-mass spectrometry, 1 H and 13 C NMR, and comparison with a chemically synthesized standard to be the expected D-Phe-L-Pro diketopiperazine. Further minimization of the two modules was not possible without a loss of transfer activity. Likewise, a mutation in a proposed active-site motif (HHXXXDG) of the condensation domain giving ProCAT(H147V), abolished the condensation reaction. These results strongly suggest the condensation domain to be involved in the catalysis of nonribosomal peptide bond formation with the histidine 147 playing a catalytic role.Microorganisms use the nonribosomal pathway to synthesize a large group of structurally diverse and often complex secondary metabolites with biological activities, including antibiotics, siderophores, biosurfactants, immunosuppressants, and antitumor and antiviral agents. These low molecular weight peptide-based compounds are synthesized by means of multifunctional enzymes, the peptide synthetases, which can recruit not only proteogenic amino acids but also a large number of unusual amino acids and hydroxy acids that form peptide and ester bonds. The incorporated constituents can be further altered by epimerization or N-methylation, and the peptide backbone can be acylated, glycosylated, or cyclized (1-3

The cyclic decapeptide antibiotic tyrocidine is produced by Bacillus brevis ATCC 8185 on an enzyme complex comprising three peptide synthetases, TycA, TycB, and TycC (tyrocidine synthetases 1, 2, and 3), via the nonribosomal pathway. However, previous molecular characterization of the tyrocidine synthetase-encoding operon was restricted to tycA, the gene that encodes the first one-module-bearing peptide synthetase. Here, we report the cloning and sequencing of the entire tyrocidine biosynthesis operon (39.5 kb) containing the tycA, tycB, and tycC genes. As deduced from the sequence data, TycB (404,562 Da) consists of three modules, including an epimerization domain, whereas TycC (723,577 Da) is composed of six modules and harbors a putative thioesterase domain at its C-terminal end. Each module incorporates one amino acid into the peptide product and can be further subdivided into domains responsible for substrate adenylation, thiolation, condensation, and epimerization (optional). We defined, cloned, and expressed in Escherichia coli five internal adenylation domains of TycB and TycC. Soluble His 6 -tagged proteins, ranging from 536 to 559 amino acids, were affinity purified and found to be active by amino acid-dependent ATP-PP i exchange assay. The detected amino acid specificities of the investigated domains manifested the colinear arrangement of the peptide product with the respective module in the corresponding peptide synthetases and explain the production of the four known naturally occurring tyrocidine variants. The K m values of the investigated adenylation domains for their amino acid substrates were found to be comparable to those published for undissected wild-type enzymes. These findings strongly support the functional integrities of single domains within multifunctional peptide synthetases. Directly downstream of the 3 end of the tycC gene, and probably transcribed in the tyrocidine operon, two tandem ABC transporters, which may be involved in conferring resistance against tyrocidine, and a putative thioesterase were found.The biosynthetic system required for the production of the cyclic decapeptide tyrocidine is one of the prototypes for a protein template-driven pathway to synthesize biologically active peptides by the nonribosomal mechanism (20,21,30,44,46). Large enzymes, called peptide synthetases, activate the amino acid constituents as aminoacyl adenylate at the expense of ATP and thioesterify them on the thiol moiety of an enzyme-attached cofactor, 4Ј-phosphopantetheine. Subsequently, activated amino acids are condensed stepwise in an aminoto carboxy-terminal direction. Previous investigations at the protein chemical level and, in particular, comparisons of several genes encoding peptide synthetases have revealed the modular architecture of this class of enzymes (4,23,28,42,44,53). One module is defined to harbor all catalytic activities to incorporate a single amino acid residue into the peptide product. The modules coincide in number with the number of incorporated amino acids and are arr...

In the biosynthesis of many macrocyclic natural products by multidomain megasynthases, a carboxy-terminal thioesterase (TE) domain is involved in cyclization and product release; however, it has not been determined whether TE domains can catalyse macrocyclization (and elongation in the case of symmetric cyclic peptides) independently of upstream domains. The inability to decouple the TE cyclization step from earlier chain assembly steps has precluded determination of TE substrate specificity, which is important for the engineered biosynthesis of new compounds. Here we report that the excised TE domain from tyrocidine synthetase efficiently catalyses cyclization of a decapeptide-thioester to form the antibiotic tyrocidine A, and can catalyse pentapeptide-thioester dimerization followed by cyclization to form the antibiotic gramicidin S. By systematically varying the decapeptide-thioester substrate and comparing cyclization rates, we also show that only two residues (one near each end of the decapeptide) are critical for cyclization. This specificity profile indicates that the tyrocidine synthetase TE, and by analogy many other TE domains, will be able to cyclize and release a broad range of new substrates and products produced by engineered enzymatic assembly lines.