The N-terminal prepeptide is required for the production of spore cortex-lytic enzyme from its inactive precursor during germination of Clostridium perfringens S40 spores

A spore cortex-lytic enzyme of Clostridium perfringens S40 is synthesized d uring sporulation as a precursor consisting of four domains. After cleavage of an N-terminal preregion and a C-terminal proregion, inactive proenzyme (termed C-35) is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination. Th e present results demonstrated that the cleaved N-terminal prepeptide remai ned associated with C-35. After the isolated complex was denatured and diss ociated in 6 M urea solution, removal of urea regenerated a prepeptide-C-35 complex which produces active enzyme when incubated with GSP. However, iso lated C-35 alone could not be activated by GSP. The prepeptide-C-35 complex was more heat stable than active enzyme. Thus, non-covalent attachment of the prepeptide to C-35 is required to assist correct folding of C-35 and to stabilize its conformation, suggesting that the prepeptide functions as an intramolecular chaperone. Recombinant proteins, which have prepeptide cova lently bonded to C-35, were processed by GSP as well as the in vivo prepept ide-C-35 complex, and the full length of the N-terminal presequence was nee ded to fulfil its role. Although the C-terminal prosequence is present as a n independent domain which is not involved in the activation process of the enzyme, it appears that the N-terminal prosequence contributes to the regu lation of enzyme activity as an inhibitor of the enzyme.