烟酰胺单核苷酸(NMN)具有抗癌和抗衰老等重要生物活性。植物中NMN含量低, 通过阻断降解途径是提高NMN含量的有效方式。植物中NMN降解途径尚不清晰。从羽衣甘蓝(Brassica oleracea var. acephala)中克隆获得8个5′-核苷酸酶候选基因, 对编码产物进行生物学信息分析, 利用大肠杆菌表达系统纯化蛋白。系统发育分析表明, 5′-核苷酸酶在植物中保守存在, 暗示其在调控核苷酸代谢中可能发挥重要作用。通过体外酶促反应研究了这些5′-核苷酸酶的催化性能, 结果表明, 羽衣甘蓝中5′-核苷酸酶BolN2、BolN5-X1和BolN6能够催化NMN降解生成烟酰胺核糖(NR)。此外, BolN2、BolN5和BolN6还能够催化烟酸单核苷酸、嘌呤和嘧啶核苷酸的水解, 具有广泛的底物适应性。研究揭示了羽衣甘蓝5′-核苷酸酶的催化特性, 为明确甘蓝中NMN的降解途径、创制高NMN含量甘蓝新种质奠定了基础。

Nicotinamide mononucleotide (NMN) has important biological activities such as anti-cancer and anti-aging. The content of NMN in plants is low, and blocking the degradation pathway is an effective way to increase the NMN content. Research on NMN degradation pathways in plants is still unclear. In this study, eight 5′-nucleotidase candidate genes were cloned from Brassica oleraceae var. acephala, the biological information of the encoded products were analyzed and the proteins were purified using the Escherichia coli expression system. Phylogenetic analysis shows that 5′-nucleotidase is conserved in plants, suggesting that it may play an important role in regulating nucleotide metabolism. In vitro enzymatic reactions were carried out to study the catalytic properties of these 5′-nucleotidases. The results showed that 5′-nucleotidase BolN2, BolN5-X1, and BolN6 from B. oleraceae var. acephala can catalyze the degradation of NMN to generate nicotinamide riboside. In addition, BolN2, BolN5, and BolN6 can also catalyze the hydrolysis of nicotinic acid mononucleotide, purine and pyrimidine nucleotides, with a wide range of substrate adaptability. This study reveals the catalytic properties of 5′-nucleotidase in B. oleraceae var. acephala, laying the foundation for exploring the degradation pathway of NMN in B. oleraceae var. acephala and creating new germplasm with high NMN content.