Mutational analysis of the pro-peptide of a marine intracellular subtilisin protease supports its role in inhibition.

ABSTRACT: Intracellular subtilisin proteases (ISPs) have important roles in protein processing during the stationary phase in bacteria. Their unregulated protein degrading activity may have adverse effects inside a cell, but little is known about their regulatory mechanism. Until now, ISPs have mostly been described from Bacillus species, with structural data from a single homolog. Here, we study a marine ISP originating from a phylogenetically distinct genus, Planococcus sp. The enzyme was successfully overexpressed in E. coli, and is active in presence of calcium, which is thought to have a role in minor, but essential, structural rearrangements needed for catalytic activity. The ISP operates at alkaline pH and at moderate temperatures, and has a corresponding melting temperature around 60?°C. The high-resolution 3-dimensional structure reported here, represents an ISP with an intact catalytic triad albeit in a configuration with an inhibitory pro-peptide bound. The pro-peptide is removed in other homologs, but the removal of the pro-peptide from the Planococcus sp. AW02J18 ISP appears to be different, and possibly involves several steps. A first processing step is described here as the removal of 2 immediate N-terminal residues. Furthermore, the pro-peptide contains a conserved LIPY/F-motif, which was found to be involved in inhibition of the catalytic activity.

SUBMITTER: Bjerga GEK 

PROVIDER: S-EPMC6220982 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature