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 during 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. The present results demonstrated that the cleaved N‐terminal prepeptide remained associated with C 35 . After the isolated complex was denatured and dissociated in 6 M urea solution, removal of urea regenerated a prepeptide–C 35 complex which produces active enzyme when incubated with GSP. However, isolated 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 covalently bonded to C 35 , were processed by GSP as well as the in vivo prepeptide–C 35 complex, and the full length of the N‐terminal presequence was needed to fulfil its role. Although the C‐terminal prosequence is present as an independent domain which is not involved in the activation process of the enzyme, it appears that the N‐terminal prosequence contributes to the regulation of enzyme activity as an inhibitor of the enzyme.