Molecular Insights into a Promiscuous Dikinase Catalyzing Monophosphorylation of Structurally Diverse Natural Polyphenols

Abstract

Dikinases function in multiple biological roles, including energy metabolism and antibiotic resistance, as shown by pyruvate phosphate dikinase and rifampin phosphotransferase. Despite their functional importance, the substrate scope and synthetic potential of dikinases remain elusive. Here, we identified a dikinase from Bacillus subtilis, namely phenolic phosphate synthetase (BsPPS), which regioselectively monophosphorylates a broad spectrum of polyphenols, including flavonoids, stilbenoids, curcuminoids, chalcones, anthraquinones, coumarins, and coumestans. BsPPS catalyzes ATP-dependent phosphorylation via a ping-pong mechanism, featuring autophosphorylation at His795 and the formation of a transient phosphohistidine intermediate. This enzyme defines a PPS-like protein family characterized by a conserved catalytic tetrad (Asp627, His629, His630, and His795) and a distinctive motif (DDHHFYIDAMLDAKAR) in the unprecedented substrate-binding domain. While most members of this family remain uncharacterized, the prevalence of PPS homologs in Bacillus spp. suggests a potential role in phenolic xenobiotic metabolism. Furthermore, PPS and its widespread homologs belong to a largely unexplored superfamily of phosphotransferases with diverse apparent functions and EC classifications. Although these enzymes share a conserved domain architecture, they display significant variation in their substrate-binding domains, indicating a vast unexplored biocatalytic toolbox. Our findings unveil a biocatalytic route for synthesizing diverse polyphenol monophosphates, opening avenues for phosphate prodrug development.