Study of the key points of lincomycin and celesticetin biosynthesis

Lincosamides form a small but important group of specialized microbial metabolites with antibiotic activity. The most important members of this group are celesticetin and clinically used lincomycin. Structurally, lincosamides are composed of an amino sugar and an amino acid connected by an amide bond. The amino acid precursors of both lincosamides remarkably differ. Proteinogenic L-proline is the precursor of celesticetin, while an unusual amino acid (2S,4R)-4-propyl- L-proline (PPL) is incorporated in the more efficient compound lincomycin. Surprisingly, both these precursors are recognized and activated for further biosynthetic steps by homologous adenylation domains CcbC and LmbC, respectively. The detailed description of this amino acid recognition and activation step, which is critical for the biological activity of the resulting compound, was the aim of the first part of this thesis. The site-directed mutagenesis of the LmbC substrate binding pocket and biochemical characterization of resulting mutants were employed to identify the residues crucial for the activation of PPL. Subsequently, we experimentally simulated the molecular evolution leading from L-proline-specific substrate binding pocket (like in CcbC) to the PPL-specific enzyme (LmbC). The substitution of only three amino acid..