Proteolytic mechanism of a novel mitochondrial and chloroplastic PreP peptidasome.

The 2.1-A-resolution crystal structure of the novel mitochondrial and chloroplastic metalloendopeptidase, At-PreP1, revealed a unique peptidasome structure, in which the two halves of the enzyme completely enfold a huge proteolytic cavity. Based on the structure, we proposed a novel mechanism for proteolysis involving hinge-bending motions, which cause the protease to open and close in response to substrate binding. We generated four double-mutants of AtPreP1 by introducing cysteines at positions where disulfide bonds can be formed in order to lock and unlock the protease and tested the activity under oxidizing and reducing conditions. The overall results support the proposed mechanism.