Structural Insights into Trehalose-6-Phosphate Phosphorylase and Its Role in Trehalose 6-Phosphate Biosynthesis via a Multienzyme Cascade

Trehalose 6-phosphate (Tre6P) is a critical metabolic signaling molecule in plants, orchestrating diverse biological processes, including stress resistance and photosynthetic efficiency. Recent advancements highlight its promising role in improving crop yield. However, the efficient synthesis of Tre6P in vitro remains a major challenge. Here, we identified a trehalose-6-phosphate phosphorylase WcTre6PPase from Weissella ceti, the enzyme catalyzes the reversible synthesis of Tre6P from β-glucose 1-phosphate (βGlc1P) and glucose 6-phosphate (Glc6P), with a strong catalytic bias for Tre6P synthesis. The crystal structure of WcTre6PPase resolved in complex with βGlc1P reveals critical molecular determinants for substrate recognition and catalytic efficiency. Binding studies demonstrate a higher affinity of WcTre6PPase for βGlc1P by 1.0 ± 0.2 mM compared to Glc6P by 24.3 ± 7.1 mM. Complementary molecular docking and dynamics simulations provide detailed insights into the catalytic mechanism. Subsequently, by integrating WcTre6PPase into a novel multienzyme cascade, comprising maltose phosphorylase, polyphosphate glucokinase, and AMP/ADP-polyphosphate phosphotransferase, and optimizing reaction conditions, we achieved a remarkable Tre6P yield of 90% from maltose. This study provides molecular insights into the function of WcTre6PPase and establishes a prospective platform for Tre6P production for agricultural applications.