Biocrystallography Group, IRCCS San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milano, Italy.
Faculty of Medicine and Surgery, Università Vita-Salute San Raffaele, Via Olgettina 58, 20132 Milano, Italy.
Int J Mol Sci. 2024 Jun 27;25(13):7032. doi: 10.3390/ijms25137032.
The URH1p enzyme from the yeast has gained significant interest due to its role in nitrogenous base metabolism, particularly involving uracil and nicotinamide salvage. Indeed, URH1p was initially classified as a nucleoside hydrolase (NH) with a pronounced preference for uridine substrate but was later shown to also participate in a Preiss-Handler-dependent pathway for recycling of both endogenous and exogenous nicotinamide riboside (NR) towards NAD+ synthesis. Here, we present the detailed enzymatic and structural characterisation of the yeast URH1p enzyme, a member of the group I NH family of enzymes. We show that the URH1p has similar catalytic efficiencies for hydrolysis of NR and uridine, advocating a dual role of the enzyme in both NAD synthesis and nucleobase salvage. We demonstrate that URH1p has a monomeric structure that is unprecedented for members of the NH homology group I, showing that oligomerisation is not strictly required for the N-ribosidic activity in this family of enzymes. The size, thermal stability and activity of URH1p towards the synthetic substrate 5-fluoruridine, a riboside precursor of the antitumoral drug 5-fluorouracil, make the enzyme an attractive tool to be employed in gene-directed enzyme-prodrug activation therapy against solid tumours.
酵母中的 URH1p 酶因其在含氮碱基代谢中的作用而受到广泛关注，特别是涉及尿嘧啶和烟酰胺的回收利用。事实上，URH1p 最初被归类为核苷水解酶（NH），对尿嘧啶底物具有明显的偏好，但后来发现它也参与了 Preiss-Handler 依赖途径，用于回收内源性和外源性烟酰胺核苷（NR）以合成 NAD+。在这里，我们对酵母 URH1p 酶进行了详细的酶学和结构表征，它是 I 组 NH 酶家族的成员。我们发现 URH1p 对 NR 和尿嘧啶的水解具有相似的催化效率，这表明该酶在 NAD 合成和核碱基回收中具有双重作用。我们证明 URH1p 具有单体结构，这在 I 组 NH 同源性家族成员中是前所未有的，表明寡聚化对于该酶家族的 N-核糖基活性不是严格必需的。URH1p 的大小、热稳定性和对合成底物 5-氟尿嘧啶的活性，5-氟尿嘧啶是抗肿瘤药物 5-氟尿嘧啶的核苷前体，使得该酶成为一种有吸引力的工具，可以用于针对实体瘤的基因定向酶前药激活治疗。
