Tricorn protease (TRI), a high molecular mass complex from the archaeon T. acidophilum, forms the core of a modular proteolytic system; upon interacting with low molecular mass factors intrinsic activities are enhanced and novel activities are generated. Here we characterize the first factor, F1, which turns out to be homologous with several bacterial proline iminopeptidases (PIPs). Surprisingly, it cleaves not only typical PIP substrates such as H-Pro-AMC, but a wide spectrum of amino acid substrates and several peptide substrates without a proline at the N-terminus. The pip gene encodes a 293 amino acid residue protein with a molecular mass of 33,487 Da. By means of site-directed mutagenesis we identified Ser105 and His271 as the active site nucleophile and proton donor, respectively. Experiments with inactive mutant PIPs indicate that the activities elicited by interacting with TRI are contributed by PIP.
