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 C35) 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 C35. After the isolated complex was denatured and dissociated in 6 M urea solution, removal of urea regenerated a prepeptide-C35 complex which produces active enzyme when incubated with GSP. However, isolated C35 alone could not be activated by GSP. The prepeptide-C35 complex was more heat stable than active enzyme. Thus, non-covalent attachment of the prepeptide to C35 is required to assist correct folding of C35 and to stabilize its conformation, suggesting that the prepeptide functions as an intramolecular chaperone. Recombinant proteins, which have prepeptide covalently bonded to C35, were processed by GSP as well as the in vivo prepeptide-C35 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.
Okamura
S
S
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Japan.
Urakami
K
K
Kimata
M
M
Aoshima
T
T
Shimamoto
S
S
Moriyama
R
R
Makino
S
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003016:Clostridium perfringens / Q000201:enzymology* / Q000254:growth & development; D017931:DNA Primers; D004789:Enzyme Activation; D006867:Hydrolases / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013171:Spores, Bacterial / Q000201:enzymology* / Q000254:growth & development
D001426:Bacterial Proteins; D017931:DNA Primers; D006867:Hydrolases; C052724:peptidoglycan-lytic enzyme, Bacillus megaterium