Structural and Functional Insights into a Previously Uncharacterized Psr LCP Protein from Streptococcus dysgalactiae subsp. dysgalactiae

There is a pressing need to develop new antibiotics targeting alternative biological pathways to effectively tackle the problem of antibiotic resistance. LytR-CpsA-Psr (LCP) proteins catalyze the attachment of cell-wall glycopolymers to peptidoglycan, a crucial step in Gram-positive bacterial envelope biogenesis and a promising target for antimicrobial development. Here we report the structural and functional characterization of a Psr-type LCP protein from Streptococcus dysgalactiae subsp. dysgalactiae (SDSD). Phylogenetic analysis places this enzyme within the Psr/LcpA clade, showing higher conservation among streptococcal homologs than with LytR-type enzymes. The AlphaFold2 model and small-angle X-ray scattering (SAXS) data reveal a conserved overall fold with subtle differences from LytR, including an elongated shape and flexible termini. Sequence analysis highlights distinctive Psr-specific residues (R235, F317, F320) that may influence substrate recognition. Enzymatic activity was confirmed using both colorimetric and nuclear magnetic resonance (NMR) assays, demonstrating conversion of ADP to AMP with maximal activity near 37°C and an apparent zero-order kinetic regime under saturating substrate conditions. The ¹H NMR assay, implemented here for the first time, provides a continuous, label-free approach to monitor LCP activity in real time. Together, these findings establish the structural conservation and functional distinctiveness of the SDSD Psr LCP domain, introduce a complementary NMR-based strategy suitable for kinetic and inhibitor studies within the LCP family and lay the groundwork for a deeper understanding of LCP-mediated processes, supporting their potential as antibiotic targets.