(182an) Energizing Enzyme Dynamics to Synthesize Ultra-High-Molecular-Weight Hyaluronic Acid in Vitro

Hyaluronic acid (HA) is a linear polysaccharide composed of repeating [GlcNAc(β1→3)GlcUA(β1→4)] units. High-molecular-weight HA (HMW-HA) exhibits notable anti-inflammatory and anti-tumor activities, and serves as an advanced biomaterial for drug delivery, tissue engineering, and beyond. In vitro enzymatic synthesis circumvents cellular limitations on polymer accumulation, providing a promising approach for efficient production of HA with increased molecular weight (Mw). Class I hyaluronan synthase (HAS) couples HA elongation and translocation in a processive manner, achieving superior control over Mw uniformity. However, in vitro enzymatic catalysis supplies a fixed driving force that becomes progressively inadequate to translocate the elongating HA chain, thereby limiting Mw.

Here, we propose a novel strategy to increase HA Mw by supplying additional energy to promote HA translocation. As proof of concept, chemical energy (ATP addition) and thermal energy (heating) are introduced. Site-specific ¹⁹F labeling and ¹⁹F NMR, ¹H NMR, molecular dynamics simulations, and site-directed mutagenesis reveal that protein motions facilitating HA translocation are enhanced upon energy supplementation. These findings provide mechanistic insights into Mw control in HA synthesis from both molecular and energy-driven perspectives. Moreover, the proposed energization strategy offers a generalizable approach for synthesizing diverse biopolymers with ultra-high Mw in vitro.