Characterization of the Saccharomyces cerevisiae cyclic nucleotide phosphodiesterase involved in the metabolism of ADP-ribose 1 '',2 ''-cyclic phosphate

ADP-ribose 1 ",2 "-cyciic phosphate (Appr>p) is produced in yeast and other eukaryotes as a consequence of tRNA splicing. This molecule is converted t o ADP-ribose 1 "-phosphate (Appr-1 " p) by the action of the cyclic nucleot ide phosphodiesterase (CPDase), Comparison of the previously cloned CPDase from Arabidopsis with proteins having related cyclic phosphodiesterase or R NA ligase activities revealed two histidine-containing tetrapeptides conser ved in these enzyme families, Using the consensus phosphodiesterase signatu re, we have identified the yeast Saccharomyces cerevisiae open reading fram e YGR247w as encoding CPDase, The bacterially expressed yeast protein, name d Cpd1p, is able to hydrolyze Appr>p to Appr-1 " p, Moreover, as with the p reviously characterized Arabidopsis and wheat CPDases, Cpd1p hydrolyzes nuc leosides 2',3'-cyclic phosphates (N>p) to nucleosides 2'-phosphates. Appare nt K-m values for Appr>p, A>p, U>p, C>p and G>p are 0.37, 4.97, 8.91, 12.18 and 14.29 mM, respectively, Site-directed mutagenesis of individual amino acids within the two conserved tetrapeptides showed that H-40 and H-150 res idues are essential for CPDase activity, Deletion analysis has indicated th at the CPD1 gene is not important for cellular viability, Likewise, overexp ression of Cpd1p had no effect on yeast growth, These results do not implic ate an important role for Appr>p or Appr-1 " p in yeast cells grown under s tandard laboratory conditions.