UDP-N-acetylglucosamine pyrophosphorylase (UAP1) catalyses the last step in eukaryotic biosynthesis of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), converting UTP and GlcNAc-1P to the sugar nucleotide. Gene disruption studies have shown that this gene is essential in eukaryotes and a possible antifungal target, yet no inhibitors of fungal UAP1 have so far been reported. Here we describe the crystal structures of substrate/product complexes of UAP1 from Aspergillus fumigatus that together provide snapshots of catalysis. A structure with UDP-GlcNAc, pyrophosphate and Mg²⁺ provides the first Michaelis complex trapped for this class of enzyme, revealing the structural basis of the previously reported Mg²⁺ dependence and direct observation of pyrophosphorolysis. We also show that a highly conserved lysine mimics the role of a second metal observed in structures of bacterial orthologues. A mechanism-inspired UTP α,β-methylenebisphosphonate analogue (meUTP) was designed and synthesized and was shown to be a micromolar inhibitor of the enzyme. The mechanistic insights and inhibitor described here will facilitate future studies towards the discovery of small molecule inhibitors of this currently unexploited potential antifungal drug target.

中文翻译：

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一种受机制启发的 UDP-N-乙酰葡糖胺焦磷酸化酶抑制剂

UDP- N-乙酰氨基葡萄糖焦磷酸化酶 (UAP1) 催化真核生物合成尿苷二磷酸-N-乙酰氨基葡萄糖 (UDP-GlcNAc)的最后一步，将 UTP 和 GlcNAc-1P 转化为糖核苷酸。基因破坏研究表明，该基因在真核生物中是必不可少的，并且是一个可能的抗真菌靶点，但迄今为止还没有关于真菌 UAP1 抑制剂的报道。在这里，我们描述了来自烟曲霉的 UAP1 底物/产物复合物的晶体结构，它们共同提供了催化快照。具有 UDP-GlcNAc、焦磷酸盐和 Mg ²⁺的结构为此类酶提供了第一个捕获的 Michaelis 复合物，揭示了先前报道的 Mg ²⁺的结构基础焦磷酸分解的依赖性和直接观察。我们还表明高度保守的赖氨酸模拟了在细菌直向同源物结构中观察到的第二种金属的作用。设计并合成了一种受机制启发的 UTP α,β-亚甲基双膦酸酯类似物 ( me UTP)，并被证明是该酶的微摩尔抑制剂。这里描述的机制见解和抑制剂将有助于未来研究发现这个目前未开发的潜在抗真菌药物靶点的小分子抑制剂。