An assay enables high-throughput screening of phosphorylation activity. • Umbelliferone enables activity readout via phosphorylation-induced fluorescence quenching. • The assay was validated with high accuracy, precision, and reproducibility. • Directed evolution using the HTS platform identified a thermostable Bs PPS variant (G145D). To date, enzymatic systems capable of phosphorylating phenolic compounds are rare, and phenolic phosphate synthetase from Bacillus subtilis ( Bs PPS) is distinguished by its ability to regioselectively phosphorylate a broad range of structurally diverse natural polyphenols, a transformation valuable for improving the aqueous solubility and bioavailability of phytochemicals. However, the lack of an efficient high-throughput screening (HTS) platform has limited its engineering and application. Here, we developed a fluorescence-based HTS assay for rapid estimation of Bs PPS activity using umbelliferone as a fluorogenic probe. Phosphorylation of umbelliferone causes pronounced fluorescence quenching, providing an inverse correlation between fluorescence intensity and catalytic activity. The assay was systematically optimized and validated, demonstrating high accuracy, precision, and reproducibility. Application to an error-prone PCR library identified Bs PPS variants with improved thermostability while maintaining catalytic activity. Overall, this assay fills a critical methodological gap and provides a practical foundation for future exploration of related phenolic compound–phosphorylating enzymes. Download: Download high-res image (211KB) Download: Download full-size image
Chen Hsu,Hsin-Ya Tsai,Sheng-Dong Chen,Chi-Fon Chang,Nan-Wei Su
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.
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