Abstract：
Excessive fructose consumption is a primary contributor to the global surges in obesity, cancer, and metabolic syndrome. Fructolysis is not robustly regulated and is initiated by ketohexokinase (KHK). In this study, we determined the crystal structure of KHK-A, one of two human isozymes of KHK, in the apo-state at 1.85 Å resolution, and we investigated the roles of residues in the fructose-binding pocket by mutational analysis. Introducing alanine at D15, N42, or N45 inactivated KHK-A, whereas mutating R141 or K174 reduced activity and thermodynamic stability. Kinetic studies revealed that the R141A and K174A mutations reduced fructose affinity by 2- to 4-fold compared to WT KHK-A, without affecting ATP affinity. Molecular dynamics simulations provided mechanistic insights into the potential roles of the mutated residues in ligand coordination and the maintenance of an open state in one monomer and a closed state in the other. Protein-protein interactome analysis indicated distinct expression patterns and downregulation of partner proteins in different tumor tissues, warranting a reevaluation of KHK\'s role in cancer development and progression. The connections between different cancer genes and the KHK signaling pathway suggest that KHK is a potential target for preventing cancer metastasis. This study enhances our understanding of KHK-A\'s structure and function and offers valuable insights into potential targets for developing treatments for obesity, cancer, and metabolic syndrome.
摘要：
过量的果糖消费是全球肥胖激增的主要原因，癌症,和代谢综合征。裂解不受强力调节,并且由酮己糖激酶(KHK)启动。在这项研究中,我们确定了KHK-A的晶体结构，KHK的两种人类同工酶之一，在阿波状态下，分辨率为1.85，我们通过突变分析研究了果糖结合口袋中残基的作用。在D15，N42或N45灭活的KHK-A处引入丙氨酸，而突变R141或K174降低了活性和热力学稳定性。动力学研究表明，与WTKHK-A相比，R141A和K174A突变可将果糖亲和力降低2至4倍，不影响ATP亲和力。分子动力学模拟提供了对突变残基在配体配位中的潜在作用以及在一个单体中维持开放状态和在另一个单体中维持封闭状态的机制见解。蛋白质-蛋白质相互作用组分析显示不同肿瘤组织中伴侣蛋白的不同表达模式和下调。保证重新评估KHK在癌症发展和进展中的作用。不同癌症基因与KHK信号通路之间的联系表明KHK是预防癌症转移的潜在靶标。这项研究增强了我们对KHK-A的结构和功能的理解，并为开发肥胖治疗的潜在目标提供了有价值的见解。癌症,和代谢综合征。
