Mammalian NADH diphosphatases of the Nudix family: cloning and characterization of the human peroxisomal NUDT12 protein
Mammalian NADH diphosphatases of the Nudix family: cloning and characterization of the human peroxisomal NUDT12 protein
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DOI：
10.1042/bj20030441
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发表时间：
2003-09-01
影响因子：
4.1
通讯作者：
McLennan, AG
中科院分区：
文献类型：
--
作者：
Abdelraheim, SR;Spiller, DG;McLennan, AG
人类 NUDT12 Nudix 水解酶已在昆虫细胞中通过杆状病毒载体表达为带有 His 标签的重组蛋白。在体外，它能有效地将 NAD(P)H 水解为 NMNH 和 AMP (2',5'-ADP)，并将二磷酸二腺苷水解为 AMP。它还对 NAD(P)(+)、ADP-核糖和三磷酸二腺苷具有活性。 NADH、NADPH和NAD(+)的K-m值分别为11、16和190μM，k(cat)值分别为11、16和10.5s(-1)。因此，与 Nudix 家族的其他 NADH 二磷酸酶一样，NUDT12 对还原的烟酰胺核苷酸具有明显的底物偏好。体外 50 mM Mn2+ 离子支持最佳活性，0.4 mM Mg2+ 时活性降低 3 倍。 NUDT12 作为绿色荧光蛋白 C 端融合体的表达表明，它通过 C 端三肽 PNL 作为新型 1 型过氧化物酶体靶向信号靶向过氧化物酶体。 PNL 的缺失导致细胞荧光扩散。此外，带有或不带有 PNL 信号的 C 端（而非 N 端）融合物会在大型、未识别的细胞质结构中积累。 NUDT12 可能调节该细胞器中氧化代谢所需的过氧化物酶体烟酰胺核苷酸辅因子的浓度。
The human NUDT12 Nudix hydrolase has been expressed in insect cells from a baculovirus vector as a His-tagged recombinant protein. In vitro, it efficiently hydrolyses NAD(P)H to NMNH and AMP (2',5'-ADP), and diadenosine diphosphate to AMP. It also has activity towards NAD(P)(+), ADP-ribose and diadenosine triphosphate. K-m values for NADH, NADPH and NAD(+) are 11, 16 and 190 muM and k(cat) values are 11, 16 and 10.5 s(-1) respectively. Thus, like other NADH diphosphatases of the Nudix family, NUDT12 has a marked substrate preference for the reduced nicotinamide nucleotides. Optimal activity was supported by 50 muM Mn2+ ions in vitro, with 3-fold lower activity at 0.4 mM Mg2+. Expression of NUDT12 as a C-terminal fusion to green fluorescent protein revealed that it was targeted to peroxisomes by the C-terminal tripeptide PNL acting as a novel type 1 peroxisomal targeting signal. Deletion of PNL resulted in diffuse cellular fluorescence. In addition, C-terminal, but not N-terminal, fusions with or without the PNL signal accumulated in large, unidentified cytoplasmic structures. NUDT12 may act to regulate the concentration of peroxisomal nicotinamide nucleotide cofactors required for oxidative metabolism in this organelle.