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Two putative nonheme iron oxygenase enzymes, KtzO and KtzP, are recombinantly expressed, characterized biochemically in vitro, and are found to stereospecifically hydroxylate the [beta]-position of glutamic acid. A method using nonhydrolyzable coenzyme A analogs is developed and used for determining the kinetic parameters for KtzO- and KtzP-catalyzed hydroxylation of glutamic acid bound to the carrier protein.

The isolation and structural characterization of capistruin, a new lasso peptide from Burkholderia thailandensis E264, is described. Mass spectrometric and NMR spectroscopic studies have shown capistruin as a new representative of lasso peptides and the heterologous production of capistruin in Escherichia coli is established.

The enzymes from the calcium-dependent antibiotics (CDA) producer Streptomyces coelicolor were used to understand the biochemical in vitro reconstitution of the 3-MeGlu biosynthetic pathways. Findings might open new ways for enzymatic synthesis of [beta]-methylated building blocks and engineering of acidic lipopeptide biosynthesis.

The general aspects of siderophore-mediated iron acquisition, iron-related pathogen-host interactions and the strategies for iron-dependent pathogen control are discussed. The inhibition of novel siderophore pathways targets is described.

The biochemical characterization of two new peptide cyclases from daptomycin and A54145 nonribosomal peptide synthetases (NRPSs) is described. The excised peptide cyclases from A54145 and daptomycin NRPSs were shown to be able to catalyze the macrocyclization of peptide thioester substrates, by applying chemoenzymatic strategy derivatives of A54145 and daptomycin were generated.

The recombinant thioesterase domain SnbDE TE of the pristinamycin I nonribosomal peptide synthetase from streptomyces pristinaespiralis is cloned, overproduced and biochemically characterized. A remarkable substrate tolerance was detected for the enzymatic cyclization including all the seven positions of the peptide backbone.

The biochemical proof is presented that the aldehyde intermediate, formed by the release of peptide from the protein template in an NAD(P)H-dependent reduction step catalyzed by the adjacent reductase, is further reduced by an aldoreductase, LgrE, in an NADPH-dependent manner to form the final product gramicidin A, N-formyl-pentadecapeptide-enthanolamine. To determine the potential use of the two reductases in the construction of hybrid NRPSs, their ability to accept a variety of different substrates in vitro is tested.

The global overview of the understanding of natural nonribosomal peptide synthesis (NRPS) and of progress in genetically engineered and chemoenzymatic synthesis of nonribosomal peptide products is provided. NRPS are highly sophisticated natural nanomachines that were optimized for the biosynthesis of compounds that cannot be produced by the ribosomal machinery and were selected during evolution for diverse structures and for broad biological activities.

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. The findings reveal that calcium-dependent antibiotic (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.

The first biochemical characterization of a recombinant cyclase of a streptomycete, the model actinomycete Streptomyces coelicolor that produces the calcium-dependent antibiotic (CDA) is reported. Results provide evidence for the significant role of N-terminal fatty acyl groups of lipopeptides in controlling the chemoselectivity, regioselectivity, and kinetics of recombinant TE mediated macrocyclization.

A study was conducted on the tetrapeptidyl-S-T4 and L-Asp-S-T5 for peptide bond formation and chain translocation by C5 to yield pentapeptidyl-S-T5. It is predicted that the peptide bond-forming condensation domains immediately downstream of E domains are D-specific for the peptidyl donor and L-specific for the aminoacyl acceptor.

Research has been conducted on cold shock response in Bacillus subtilis. Results demonstrate that B. subtilis two-component system encoded by desKR operon is important for cold-induced expression of lipid-modifying desaturase.

Experiments were conducted, which show that a C-terminal reductase domain of the respective nostocyclopeptide (ncp) nonribosomal peptides (NRPs) catalyzes the reductive release of the mature peptide chain from the synthetase and thereby triggers the spontaneous formation of the imino head-to-tail linkage. The experiments also provided proof that the ncp molecular self-assembly is based on the intrinsic conformation of the linear heptapeptide aldehyde and strongly depends on the nature of the C- and N-terminal amino acids.

A study was conducted to investigate the catalytic function of the putative formylation domain and the mode of action of N-formylation in linear gramicidin biosynthesis in order to test its interaction with the natural neighboring domains and the timing of the initiation reaction of the nonribosomal peptide synthetases (NRPS) assembly line. The experimental data indicate that the F domain is essential for the initiation of linear gramicidin biosynthesis.

A new approach to obtain hybrid nonribosomal peptide synthetases (NRPSs) is used to show that the deletion of an entire module in an NRPS assembly line caused the secretion of the predicted peptide antibiotic variant with a decreased ring size. It is concluded that the surgery in the linkers between entire modules seems to be a robust approach to rationally design NRPSs in nonribosomal peptide antibiotic producer strains.