engleski

Exploring the role of A9 conserved motif in the adenylation domain of tyrocidine synthetase 1 from Bacillus brevis

Nonribosomal peptide synthetases (NRPSs) are modular proteins responsible for the production of peptide natural products, many of which have antibiotic activity. The modular nature of NRPS makes them suitable targets for engineering through domain or module rearrangements. In practice, the ability to obtain functional enzymes depends on the extent to which the protein-protein interactions necessary for catalytic activity of the engineered NRPS assembly have been preserved. High-resolution NMR and crystal structures of NRPS domains, didomains and even an entire module obtained during last several years greatly expanded our understanding of intra- and intermodular protein interactions in NRPS systems. Distinct protein-interaction surfaces were reported mainly for the peptidyl carrier protein (PCP) domain ; however, several attempts to map protein interaction surfaces on adenylation (A) domain failed to pinpoint a particular region of the protein. Our search for the putative surface on the A domain, involved in protein interaction with the PCP domain was based on sequence analysis and homology modeling. We examined all the available structural data not only from NRPS A domains, but also of the other members of adenylate-forming superfamily, and hypothesized that the region of A domain assigned as A9 core motif could be of importance for A and PCP domain interaction. When analyzing homology models build for the protein conformation in the first half-reaction (amino acid activation) and during the second half-reaction (transfer of the activated amino acid to the 4’-phosphopantetheine arm of the adjacent PCP domain), this region seems to position itself favorably for the interaction with PCP domain during the second half-reaction. To test this hypothesis, we created a set of mutant proteins, in which single amino acid exchanges were introduced into the A9 core motif of A domain from tyrocidine synthetase 1, 484LPAYMLP. The mutant proteins were expressed and purified from E. coli as His-tagged proteins. The influence of mutation in A9 motif was examined by assaying enzymatic activity (with or without the acceptor of activated amino acid in the second-half reaction), susceptibility to proteolysis, and fluorescence properties of the mutant enzymes. The results obtained indicate that, while the A9 core motif is not indispensable for the activation of amino acid, it contributes to the structural stabilization of the protein and might play an important role for the transfer of activated amino acid from the adenylation onto the PCP domain.

adenylation domain; conserved motif; nonribosomal peptide synthetases; tyrocidine synthetase 1

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