engleski

Exploring the role of a conserved motif in the adenylation domain of a non-ribosomal peptide synthetase from Bacillus brevis

Non-ribosomal peptide synthetases (NRPS) are molecular machines that synthesize peptide-based natural products, many of which are important pharmaceuticals with antibiotic, immunosuppressor and antitumor activities. A typical NRPS assembly line consists of a number of catalytic modules, each responsible for the incorporation of a single amino acid into the growing peptide product. Modules themselves comprise distinct catalytic domains, such as the adenylation domain (A) that, firstly, selects a specific amino acid monomer to be activated by ATP and, secondly, transfers it to the phosphopantetheinyl arm of the adjacent thiolation domain. Recent biochemical and structural evidence support the idea that the A domain uses a rotational movement of its two subdomains to adapt a single active site for the two partial reactions in the catalytic cycle. It has long been recognized that A domains share a set of 10 conserved motifs (A1-A10), most of which where assigned a particular role in substrate binding and/or catalysis. To our knowledge, this is the first study aimed at elucidating the role of the A9 motif. By analyzing homology models of the A domain from tyrocidine synthetase 1 from Bacillus brevis, it could be observed that during the rotational movement of the subdomains, the A9 motif drastically changes its position relative to the active site. The role of the conserved amino acid residues in this region and its importance for the two partial reactions of the catalytic cycle was examined by site-directed mutagenesis and biochemical characterization of mutant enzymes.

adenylation domain; peptide synthetase

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