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Substrate-induced conformational changes of the adenylation domain from Tyrocidine synthetase 1 probed by intrinsic Trp fluorescence

Nonribosomal peptide synthetases (NRPS) are large multifunctional enzymes that catalyse the synthesis of nonribosomal peptides (NRP) known for a broad range of pharmaceutical properties such as antimicrobial, immunosuppressive and antitumor activity. With overwhelming reports of bacterial resistance, it is of fundamental importance to gain more insight into structural and functional properties of these megastructures. Biosynthesis of NRP starts with the adenylation (A) domain that activates a specific amino acid substrate in form of aminoacyl-adenylate with concomitant release of pyrophosphate. The activated amino acid is subsequently transferred via the peptidyl carrier protein (PCP) domain to the neighbouring active site of the condensation (C) domain that finally catalyses peptide bond formation. Crystallographic studies on various NRPS A-domains showed that these enzymes undergo extensive structural rearrangements during the catalytic cycle. Two catalytically distinct conformations are reported for A-domains: the first being implied in adenylate formation, and the second in transfer of the activated amino acid onto the PCP-domain. Here we report the first extensive study on fluorescence properties of a representative A-domain from tyrocidine synthetase 1 (TycA-A). TycA-A comprises five potentially fluorescent Trp residues designated W227, W301, W323, W376 and W406, respectively. Individual Trp accessibility surface area (ASA) and their structural position were assessed based on a structural model of the TycA-A protein in both conformations. To resolve which Trp contributes most to the emission spectrum of TycA-A, single point mutants bearing Trp to Phe substitutions were constructed. Mutant proteins were tested for thermal and structural stability using the thermal shift assay and limited proteolysis. Acrylamide quenching was employed to probe accessibility of individual Trp residues upon substrate binding to mutant and wild type protein, respectively. Our results show that among five Trp residues only W227 reports to substrate binding. Conformational changes upon non-cognate amino acid binding was also probed by acrylamide quenching in a mutant protein bearing only W227. This showed that the extent of quenching is more evident with non-cognate substrates, indicating a suboptimal overall conformation with concomitant hydrolysis of the aminoacyl-adenenylate.

Adenylation domain; Nonribosomal peptide synthetases; Conformational changes; fluorescence quenching

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