Department of Chemistry, UiT the Arctic University of Norway, PO Box 6050, Stakkevollan, 9037, Langnes, Tromsø, Norway.
Department of Physics, Chemistry and Biology, Linköping University, 581 83, Linköping, Sweden.
Sci Rep. 2024 Oct 30;14(1):26081. doi: 10.1038/s41598-024-76548-x.
Structural- and functional heterogeneity, as well as allosteric regulation, in homo-monomeric enzymes is a highly active area of research. One such enzyme is human nuclear-associated deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), which has emerged as an interesting drug target in combination therapy with traditional nucleotide analogue treatment of cancer. We report, for the first time, a full structural dynamics study of human dUTPase by NMR. dUTPase has been investigated in terms of structural dynamics in its apo form, in complex with the modified substrate resistant to hydrolysis, 2'-deoxyuridine 5'-α,β-imido-triphosphate (dUpNHpp), as well as the product, 2'-deoxy-uridine-monophosphate (dUMP). The apo form of the enzyme displayed slow dynamics in the milli- to microsecond regime in relaxation dispersion experiments, which was further slowed down to observable heterogeneity upon substrate-analogue binding. The results suggest that the non-hydrolysable substrate-analogue traps the enzyme in the conformational isomerization step that has been previously suggested to be part of the enzyme catalysis kinetics cycle. The observed heterogeneity fits well with the pattern expected to emerge from the suggested kinetic model, and no evidence for homotropic allosterism was found. The heatmaps of the slow dynamics, chemical shift perturbation upon substrate binding and conserved regions of the enzyme sequence all displayed a similar pattern, which suggests that the structural dynamics is finely tuned and important for the biological function of the enzyme for binding, conformational shift, catalysis and substrate release.
同源单体酶的结构和功能异质性以及变构调节是一个非常活跃的研究领域。人核相关脱氧尿嘧啶 5'-三磷酸核苷水解酶（dUTPase）就是这样一种酶，它作为一种有前途的药物靶点，与传统核苷酸类似物联合治疗癌症。我们首次通过 NMR 报道了人 dUTPase 的完整结构动力学研究。从结构动力学的角度研究了apo 形式的 dUTPase、与水解抗性修饰底物 2'-脱氧尿苷 5'-α,β-亚氨基三磷酸（dUpNHpp）形成的复合物形式，以及产物 2'-脱氧尿苷-单磷酸（dUMP）形式的 dUTPase。酶的apo 形式在弛豫分散实验中表现出毫微秒到微秒范围内的缓慢动力学，而在底物类似物结合后进一步减慢到可观察的异质性。结果表明，非水解底物类似物将酶固定在构象异构化步骤中，这被认为是酶催化动力学循环的一部分。观察到的异质性与从建议的动力学模型中得出的模式非常吻合，并且没有发现同型变构作用的证据。缓慢动力学的热图、底物结合后化学位移的变化以及酶序列的保守区域都显示出相似的模式，这表明结构动力学是精细调节的，对于酶的结合、构象转变、催化和底物释放的生物学功能非常重要。
