Mammalian NADH diphosphatases of the Nudix family: cloning and characterization of the human peroxisomal NUDT12 protein.
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 microM 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 microM Mn(2+) ions in vitro, with 3-fold lower activity at 0.4 mM Mg(2+). 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.
Abdelraheim
Salama R
SR
Cell Regulation and Signalling Group, School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, UK.
Spiller
David G
DG
McLennan
Alexander G
AG
D000595:Amino Acid Sequence; D000679:Amygdala / Q000201:enzymology; D000818:Animals; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000302:isolation & purification; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D020675:Peroxisomes / Q000737:chemistry / Q000201:enzymology*; D011755:Pyrophosphatases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D018411:Spodoptera; D013379:Substrate Specificity; D000096964:Nudix Hydrolases
D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011755:Pyrophosphatases; D011994:Recombinant Proteins; C478128:NUDT12 protein, human; D000096964:Nudix Hydrolases; C021899:nucleotide pyrophosphatase