Probing substrate recognition of bacterial lipoprotein signal peptidase using FRET reporters

Lipoprotein signal peptidase (Lsp) is a transmembrane aspartic acid protease with a pivotal role in the bacterial lipoprotein maturation pathway. Despite the universal use of Lsp across the Bacterial Kingdom and its potential as an antibiotic target, the substrate recognition patterns of Lsp are poorly understood. Here, we investigated the substrate recognition and biochemical properties of Lsp from Gram⁻ (Escherichia coli) and Gram⁺ (Streptococcus pyogenes) bacteria, using synthetic peptide-based FRET reporters. Didecanoyl glycerol was found to be an optimal lipid length, and Lsp demonstrated exclusive enantio-selectivity for the (R)-form of the diacylglycerol. Our study will facilitate the iterative optimization of in vitro Lsp assays, as well as provide the first chemical interrogation into the substrate scope of Lsp.