An acetyl-dependent proofreading mechanism governs synthetic fidelity in Phytophthora sojae chitin synthase

Chitin is an essential structural polysaccharide in fungal and oomycete cell walls, providing mechanical strength and integrity due to its crystalline, insoluble nature. Chitin synthase (CHS) is the key enzyme responsible for its biosynthesis, orchestrating the sequential steps of substrate binding, hydrolysis, glycosidic bond formation, and polymer translocation. However, the molecular mechanism that governs its synthetic fidelity remains unclear. To address this, we investigated whether Phytophthora sojae chitin synthase 1 (PsChs1) can utilize UDP-glucosamine (UDP-GlcN), a deacetylated substrate analog that closely resembles the natural substrate. Biochemical assays revealed that while PsChs1 binds and hydrolyzes UDP-GlcN, it fails to polymerize it. To uncover the structural basis of this stringent discrimination, we determined the cryo-EM structure of PsChs1 in complex with UDP-GlcN and GlcN. The structure, supported by molecular dynamics simulations, captures a non-productive state. In this state, a conserved arginine (Arg538) experiences electrostatic repulsion from the C2 ammonium group of UDP-GlcN. Concurrently, the loss of acetyl-dependent interactions displaces the 4'-OH group of the acceptor GlcN beyond the distance required for nucleophilic attack. Together, these findings reveal an acetyl-dependent proofreading mechanism that dictates the strict synthetic fidelity of chitin synthase.