Chitin, the most abundant aminopolysaccharide in nature, is an extracellular polymer consisting of N-acetylglucosamine (GlcNAc) units¹. The key reactions of chitin biosynthesis are catalysed by chitin synthase^2,3,4, a membrane-integrated glycosyltransferase that transfers GlcNAc from UDP-GlcNAc to a growing chitin chain. However, the precise mechanism of this process has yet to be elucidated. Here we report five cryo-electron microscopy structures of a chitin synthase from the devastating soybean root rot pathogenic oomycete Phytophthora sojae (PsChs1). They represent the apo, GlcNAc-bound, nascent chitin oligomer-bound, UDP-bound (post-synthesis) and chitin synthase inhibitor nikkomycin Z-bound states of the enzyme, providing detailed views into the multiple steps of chitin biosynthesis and its competitive inhibition. The structures reveal the chitin synthesis reaction chamber that has the substrate-binding site, the catalytic centre and the entrance to the polymer-translocating channel that allows the product polymer to be discharged. This arrangement reflects consecutive key events in chitin biosynthesis from UDP-GlcNAc binding and polymer elongation to the release of the product. We identified a swinging loop within the chitin-translocating channel, which acts as a ‘gate lock’ that prevents the substrate from leaving while directing the product polymer into the translocating channel for discharge to the extracellular side of the cell membrane. This work reveals the directional multistep mechanism of chitin biosynthesis and provides a structural basis for inhibition of chitin synthesis.

甲壳素是自然界中最丰富的氨基多糖，是一种由N-乙酰氨基葡萄糖 (GlcNAc) 单元组成的细胞外聚合物¹ 。几丁质生物合成的关键反应由几丁质合酶^2,3,4催化，这是一种膜整合糖基转移酶，可将 GlcNAc 从 UDP-GlcNAc 转移到不断增长的几丁质链。然而，这一过程的确切机制尚未阐明。在这里，我们报告了来自毁灭性大豆根腐病致病卵菌大豆疫霉（ Ps Chs1）的几丁质合酶的五种冷冻电子显微镜结构。它们代表了酶的 apo、GlcNAc 结合、新生几丁质寡聚物结合、UDP 结合（合成后）和几丁质合酶抑制剂尼可霉素 Z 结合状态，提供了几丁质生物合成及其竞争性抑制的多个步骤的详细视图。该结构揭示了甲壳素合成反应室，该反应室具有底物结合位点、催化中心和允许产物聚合物排出的聚合物易位通道的入口。这种排列反映了几丁质生物合成中从 UDP-GlcNAc 结合和聚合物延伸到产物释放的连续关键事件。我们在几丁质易位通道内发现了一个摆动环，它充当“门锁”，防止底物离开，同时引导产物聚合物进入易位通道以排出到细胞膜的细胞外侧。该工作揭示了几丁质生物合成的定向多步机制，并为抑制几丁质合成提供了结构基础。