Cytosolic 5¢-nucleotidase/phosphotransferase specific for 6-hydroxypurine monophosphate derivatives (cN-II), belongs to a class of phosphohydrolases that act through the formation of an enzyme–phosphate intermediate. Sequence alignment with members of the P-type ATPases/L-2-halo-acid dehalogenase superfamily identified three highly con-served motifs in cN-II and other cytosolic nucleotidases. Mutagenesis studies at specific amino acids occurring in cN-II conservedmotifswereperformed.