Mouse Nudt13 is a Mitochondrial Nudix Hydrolase with NAD(P)H Pyrophosphohydrolase Activity

Abstract

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 _m and k _cat values for NADH were 0.34 mM and 7 s^-1 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.

Keywords: Mitochondria; NADH; Nucleotide metabolism; Nudix; Pyrophosphatase.

Fig. 1

Expression of Nudt13 in High Five™ cells. High Five™ cells were infected with recombinant Nudt13 virus at a MOI of 10 for 48 h. Samples were analysed by SDS-PAGE (15% w/v) and stained with Coomassie Blue. a Lane 1 protein standards: bovine serum albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde 3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), soybean trypsin inhibitor (20 kDa) and α-lactalbumin (14.2 kDa); lane 2 control uninfected High Five™ cells; lane 3 high Five™ cells infected with Nudt13 virus for 48 h; lane 4 purified Nudt13. b Immunoblot analysis of Nudt13 (the same cells as in a, lane 3) using His.Tag monoclonal antibody

Fig. 2

Determination of reaction products of NADH and NADPH hydrolysis by Nudt13. The reaction mixtures contained 0.25 mM NADH (a, b) or NADPH (c, d) and were incubated at 37 °C for 10 min without (a, c) or with (b, d) 0.25 µg Nudt13 and the products were separated by HPLC. Absorbance at 259 nm (lines); absorbance at 340 nm (dashed lines). Products were identified by comparison to authenticated standards

Fig. 3

Subcellular localization of Nudt13 by fluorescence confocal microscopy. a EGFP fluorescence of HeLa cells transfected with pNudt13-EGFP; b EGFP fluorescence of HeLa cells transfected with pEGFP-Nudt13; c EGFP fluorescence of HeLa cells transfected with the pEGFP-C2 vector; d EGFP fluorescence of a sample cell transfected with pNudt13-EGFP; e red fluorescence of the same cell in d stained with MitoTracker red CM-H₂XRos; f superimposition of d and e on the bright field picture of the same cell