Hyaluronan Synthase Encodes Alternating Substrate Specificity by Allosteric Gating and Electrostatic Selection

Hyaluronic acid (HA) is a critical polysaccharide in the extracellular matrix, characterized by alternating β-1,3-N-acetylglucosamine and β-1,4-glucuronic acid units. It is synthesized by Class I hyaluronan synthase (HAS) within a single active site pocket, yet the molecular mechanisms underlying the alternating substrate specificity remain unresolved. Here, we demonstrated in vitro HA synthesis using Streptococcus equisimilis HAS, which maintains its alternating sequence irrespective of the substrate ratios and concentrations. Molecular dynamics simulations and mutagenesis experiments revealed that the alternating uptake of substrates is jointly determined by HA-substrate interactions and distal enzyme dynamics of the transmembrane and substrate tunnels. The lingering HA termini in the active site pocket favor HAS binding with alternating substrates through Coulombic repulsion and steric interactions. Moreover, the dynamic C-terminal loop at the substrate tunnel entrance reduces the transport distance for alternating substrates. This study provides critical molecular insights into multisubstrate specificity within a single active site pocket during polysaccharide synthesis, establishing a fundamental regulatory mechanism for sequence-defined biopolymer synthesis.