Cloning, expression and characterisation of a human Nudix hydrolase specific for adenosine 5′-diphosphoribose (ADP-ribose)

The human NUDT9 gene has been mapped to 4q22 and shown to give rise to two alternatively spliced mRNAs, NUDT9α and NUDT9β, that encode a member of the Nudix hydrolase family. Both transcripts were readily detected in heart and skeletal muscle and also in liver, kidney and pancreas. NUDT9α protein was expressed in Escherichia coli and shown specifically to hydrolyse ADP-ribose and IDP-ribose to the corresponding nucleoside 5′-monophosphates and ribose 5-phosphate. No other nucleotide substrates were hydrolysed significantly. NUDT9α was inhibited by fluoride and by N-acetyl-p-benzoquinoneimine and had K_m and k_cat values of 180 μM and 8 s^-1 respectively with ADP-ribose as substrate. The full-length 39.1 kDa NUDT9α has a potential mitochondrial leader sequence, which would give rise to a mature 34.2 kDa mitochondrial protein. Apart from the high K_m value, the properties of NUDT9α are close to those of the known mammalian 40 kDa cytoplasmic ADPRibase-1 and 35 kDa mitochondrial ADPRibase-m. However, any relationship between the NUDT9 species and the previously reported ADPRibases remains to be established.