O'Brien John H, Kadirvelraj Renuka, Tseng Po-Sen, Ross-Kemppinen Nolan, Crich David, Walsh Richard M, Wood Zachary A
Department of Biochemistry & Molecular Biology, University of Georgia, Athens, Georgia 30602, United States.
Department of Pharmaceutical and Biomedical Sciences, Department of Chemistry, and Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, United States.
Biochemistry. 2025 Jan 7;64(1):92-104. doi: 10.1021/acs.biochem.4c00555. Epub 2024 Dec 16.
Human UDP-glucose dehydrogenase (hUGDH) catalyzes the oxidation of UDP-glucose into UDP-glucuronic acid, an essential substrate in the Phase II metabolism of drugs. hUGDH is a hexamer that exists in an equilibrium between an active (E) state and an inactive (E) state, with the latter being stabilized by the binding of the allosteric inhibitor UDP-xylose (UDP-Xyl). The allosteric transition between E and E is slow and can be observed as a lag in progress curves. Previous analysis of the lag suggested that unliganded hUGDH exists mainly as E, but two unique crystal forms suggest that the enzyme favors the E state. Resolving this discrepancy is necessary to fully understand the allosteric mechanism of hUGDH. Here, we used cryo-EM to show that recombinant hUGDH expressed in copurifies with UDP-4-keto-xylose (UX4O), which mimics the UDP-Xyl inhibitor and favors the E state. Cryo-EM studies show that removing UX4O from hUGDH shifts the ensemble to favor the E state. This shift is consistent with progress curve analysis, which shows the absence of a lag for unliganded hUGDH. Inhibition studies show that hUGDH has similar affinities for UDP-Xyl and UX4O. The discovery that UX4O inhibits allosteric hUGDH suggests that UX4O may be the physiologically relevant inhibitor of allosteric UGDHs in bacteria that do not make UDP-Xyl.
人尿苷二磷酸葡萄糖脱氢酶（hUGDH）催化尿苷二磷酸葡萄糖氧化为尿苷二磷酸葡萄糖醛酸，这是药物Ⅱ相代谢中的一种必需底物。hUGDH是一种六聚体，以活性（E）状态和非活性（E）状态之间的平衡形式存在，后者通过变构抑制剂尿苷二磷酸木糖（UDP-Xyl）的结合而稳定。E和E之间的变构转变很慢，可以在进程曲线中观察到滞后现象。先前对滞后现象的分析表明，未结合配体的hUGDH主要以E形式存在，但两种独特的晶体形式表明该酶更倾向于E状态。解决这一差异对于全面理解hUGDH的变构机制至关重要。在这里，我们使用冷冻电镜表明，在共纯化过程中表达的重组hUGDH与尿苷二磷酸4-酮木糖（UX4O）结合，UX4O模拟UDP-Xyl抑制剂并倾向于E状态。冷冻电镜研究表明，从hUGDH中去除UX4O会使整体向有利于E状态的方向转变。这种转变与进程曲线分析一致，该分析表明未结合配体的hUGDH不存在滞后现象。抑制研究表明，hUGDH对UDP-Xyl和UX4O具有相似的亲和力。UX4O抑制变构hUGDH的发现表明，UX4O可能是不产生UDP-Xyl的细菌中变构UGDH的生理相关抑制剂。
