Abdelraheim Salama R, Spiller David G, McLennan Alexander G
Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, UK.
Department of Biochemistry, Faculty of Medicine, Minia University, Minia, 61519, Egypt.
Protein J. 2017 Oct;36(5):425-432. doi: 10.1007/s10930-017-9734-x.
The mammalian NUDT13 protein possesses a sequence motif characteristic of the NADH pyrophosphohydrolase subfamily of Nudix hydrolases. Due to the persistent insolubility of the recombinant product expressed in Escherichia coli, active mouse Nudt13 was expressed in insect cells from a baculovirus vector as a histidine-tagged recombinant protein. In vitro, it efficiently hydrolysed NADH to NMNH and AMP and NADPH to NMNH and 2',5'-ADP and had a marked preference for the reduced pyridine nucleotides. Much lower activity was obtained with other nucleotide substrates tested. K and k values for NADH were 0.34 mM and 7 s respectively. Expression of Nudt13 as an N-terminal fusion to green fluorescent protein revealed that it was targeted exclusively to mitochondria by the N-terminal targeting peptide, suggesting that Nudt13 may act to regulate the concentration of mitochondrial reduced pyridine nucleotide cofactors and the NAD(P)/NAD(P)H ratio in this organelle and elsewhere. Future studies of the enzymology of pyridine nucleotide metabolism in relation to energy homeostasis, redox control, free radical production and cellular integrity should consider the possible regulatory role of Nudt13.
哺乳动物的NUDT13蛋白具有Nudix水解酶家族中NADH焦磷酸水解酶亚家族的序列基序特征。由于在大肠杆菌中表达的重组产物持续不溶，活性小鼠Nudt13在昆虫细胞中通过杆状病毒载体表达为带有组氨酸标签的重组蛋白。在体外，它能有效地将NADH水解为NMNH和AMP，将NADPH水解为NMNH和2',5'-ADP，并且对还原型吡啶核苷酸有明显偏好。对于所测试的其他核苷酸底物，活性要低得多。NADH的K 和k 值分别为0.34 mM和7 s。将Nudt13作为绿色荧光蛋白的N端融合蛋白表达表明，它通过N端靶向肽专门靶向线粒体，这表明Nudt13可能在调节线粒体还原型吡啶核苷酸辅因子的浓度以及该细胞器和其他地方的NAD(P)/NAD(P)H比值方面发挥作用。未来关于吡啶核苷酸代谢酶学与能量稳态、氧化还原控制、自由基产生和细胞完整性的研究应考虑Nudt13可能的调节作用。
