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

Salama R Abdelraheim,Alexander G Mclennan,David G Spiller

doi:10.1007/s10930-017-9734-x

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. Km and kcat 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.

Highlights

Mammalian genomes typically possess 20–25 genes for members of the Nudix superfamily
This is based on the presence of a putative N-terminal mitochondrial targeting sequence and the sequence motif “SQPWPFPxS” that is found in all characterized NADH pyrophosphohydrolases downstream of the catalytic nudix box [3]
Nudt13 showed a high degree of specificity towards NADH and NADPH compared with other related nucleotides when assayed at a fixed concentration of 0.5 mM

Summary

Introduction

Mammalian genomes typically possess 20–25 genes for members of the Nudix superfamily. Several Nudix hydrolases have broad substrate specificities in vitro, making it difficult to ascertain their functions in vivo [2]. This uncertainty is compounded by the common misannotation of uncharacterized Nudix proteins in online databases as, for example, ADP-ribose pyrophosphatases or antimutator 8-oxo-dGTPases based on sequence similarities to well characterized proteins with these activities; experimental characterization is important. Most mammalian nudix proteins have been well studied, but a few, such as NUDT13, have not. NUDT13 is annotated in some databases as a mitochondrial NADH pyrophosphohydrolase This is based on the presence of a putative N-terminal mitochondrial targeting sequence and the sequence motif “SQPWPFPxS” that is found in all characterized NADH pyrophosphohydrolases downstream of the catalytic nudix box [3]. A mitochondrial location has been suggested from a proteomic study

Methods

Results

Conclusion