Hyaluronan (HA) is an essential component of the vertebrate extracellular matrix. It is a heteropolysaccharide of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) reaching several megadaltons in healthy tissues. HA is synthesized and translocated in a coupled reaction by HA synthase (HAS). Here, structural snapshots of HAS provide insights into HA biosynthesis, from substrate recognition to HA elongation and translocation. We monitor the extension of a GlcNAc primer with GlcA, reveal the coordination of the uridine diphosphate product by a conserved gating loop and capture the opening of a translocation channel to coordinate a translocating HA polymer. Furthermore, we identify channel-lining residues that modulate HA product lengths. Integrating structural and biochemical analyses suggests an avenue for polysaccharide engineering based on finely tuned enzymatic activity and HA coordination.

透明质酸 （HA） 是脊椎动物细胞外基质的重要组成部分。它是 N-乙酰氨基葡萄糖 （GlcNAc） 和葡萄糖醛酸 （GlcA） 的杂多糖，在健康组织中达到数兆道尔顿。HA 在 HA 合酶 （HAS） 的偶联反应中合成和转位。在这里，HAS 的结构快照提供了对 HA 生物合成的见解，从底物识别到 HA 伸长和易位。我们监测 GlcNAc 引物与 GlcA 的延伸，揭示尿苷二磷酸产物通过保守的门控环的配位，并捕获易位通道的开放以协调易位 HA 聚合物。此外，我们鉴定了调节 HA 产物长度的通道衬里残基。整合结构和生化分析表明了一种基于微调酶活性和 HA 配位的多糖工程途径。