A Thr/Ser-centered phosphate-binding triad of phosphate-binding protein SphX from Synechocystis.

Coordinating phosphate acquisition with overall nutritional status is critical for cyanobacterial survival in oligotrophic environments, yet the molecular basis for substrate selectivity in phosphate-binding proteins (PBPs) remains incompletely understood. Here we reveal two evolutionarily distinct PBP architectures in Synechocystis sp. PCC 6803 through integrative structural and biochemical analyses. High-resolution crystal structures (1.76-1.9 Å) of PstS1 and SphX bound to inorganic phosphate (Pi) demonstrate that D-type PBPs utilize a conserved aspartate-mediated low-barrier hydrogen bond (LBHB) for high-affinity Pi binding (nanomolar Kd), whereas S-type SphX employs a previously uncharacterized Asp-Arg-Thr catalytic triad with reduced affinity (micromolar Kd). Despite forming comparable hydrogen bonding networks (14 versus 15 bonds), SphX exhibits 26-fold lower Pi affinity due to threonine-mediated proton acceptance replacing the LBHB mechanism. Systematic mutagenesis confirms the functional importance of each triad residue and indicates partial compensation by neighboring residues. Phylogenetic analysis demonstrates that these architectural variants are conserved across cyanobacterial lineages, representing adaptive solutions to diverse nutrient-limited environments. Our findings establish a mechanistic framework for understanding PBP functional diversification and provide molecular insights relevant to optimizing cyanobacterial productivity in biotechnology applications.