Relationship between activating and editing functions of the adenylation domain of apo-tyrocidin synthetase 1 (apo-TY1).
Tyrocidine synthetase 1 (TY1), the initial monomodular constituent of the tyrocidine biosynthetic system, exhibits relaxed substrate specificity, however an efficient editing of the mis-activated amino acid provides for fidelity of product formation. We chose to analyse the consequence of single amino acid substitutions, in the amino acid activation site of apo-TY1, on the editing functions of the enzyme. Discrimination between L-Phe and D-Phe by apo-TY1 depends primarily on the editing reaction. Distraction of unnatural amino acid substrates, such as L-PheSer, implies that editing is not designated to select a specific mis-activated amino acid, but instead to discriminate all mis-activated amino acid analogues. It was shown that active site residues which interact with the adenylate are essential for both activation and editing. Substitution of Lys186 with arginine substantially reduces the editing capacity of the protein. Loss of amino acid discrimination ability by the apo-K186T and apo-R416T mutant proteins suggests a role of active site residues in maintaining the structural determinants for substrate selection. Inadequate conformational changes, induced by non-cognate amino acid substrates, promote ATP breakdown yielding P(i) and ADP. Replacement of residue Lys186 or Arg416 enhances ATP hydrolysis implying a role in binding or adjusting of the triphosphate chain for adenylate formation and pyrophosphate cleavage.