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

Abstract

ADP-ribose 1",2"-cyclic phosphate (Appr>p) is produced in yeast and other eukaryotes as a consequence of tRNA splicing. This molecule is converted to ADP-ribose 1"-phosphate (Appr-1"p) by the action of the cyclic nucleotide phosphodiesterase (CPDase). Comparison of the previously cloned CPDase from Arabidopsis with proteins having related cyclic phosphodiesterase or RNA ligase activities revealed two histidine-containing tetrapeptides conserved in these enzyme families. Using the consensus phosphodiesterase signature, we have identified the yeast Saccharomyces cerevisiae open reading frame YGR247w as encoding CPDase. The bacterially expressed yeast protein, named Cpd1p, is able to hydrolyze Appr>p to Appr-1"p. Moreover, as with the previously characterized Arabidopsis and wheat CPDases, Cpd1p hydrolyzes nucleosides 2',3'-cyclic phosphates (N>p) to nucleosides 2'-phosphates. Apparent 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)residues are essential for CPDase activity. Deletion analysis has indicated that the CPD1 gene is not important for cellular viability. Likewise, overexpression of Cpd1p had no effect on yeast growth. These results do not implicate an important role for Appr>p or Appr-1"p in yeast cells grown under standard laboratory conditions.

Biophysical Characterization of Cyclic Nucleotide Phosphodiesterases

“…This hypothesis was supported by the crystal structure (11,12) and a mutagenesis study with CPDase (S. cerevisiae) (4). Based on the current results, it is absolutely clear that these four residues are essential for the catalytic reaction since variation in either one of the four positions leads to almost complete reduction of interaction enthalpy.…”

“…Cyclic nucleotide phosphodiesterases (CPDases) characterized in wheat [L8], Arabidopsis thaliana (1,2), and yeast (1,3,4), constitute one group within a large family of proteins that includes at least four different classes of enzymes having cyclic nucleotide phosphodiesterase or related activities. On the primary structure level, enzymes of this family consist of chains containing approximately 200 amino acids and share a tandem signature motif, H-⌽-T/S-⌽, with ⌽ being a hydrophobic residue (L, V, I) (4).…”

“…On the primary structure level, enzymes of this family consist of chains containing approximately 200 amino acids and share a tandem signature motif, H-⌽-T/S-⌽, with ⌽ being a hydrophobic residue (L, V, I) (4).…”

“…A partially purified enzyme from yeast was found to use specifically Appr Ͼ p, but not N Ͼ p, as a substrate (1). However, the CPDase gene in Saccharomyces cerevisiae has been identified recently and it was shown that the recombinant yeast protein hydrolyses both Appr Ͼ p and N Ͼ p (3,4). It seems likely that the specificity of this enzyme is controlled by some additional proteins or other factors.…”

“…The active site is comprised of the tandem signature motif (residues His42, Thr44, His119, and Ser121) located in a water-filled cavity found within the core of the protein. A mutation study with the yeast CPDase revealed that the presence of the histidine residues of the signature motif is essential for the catalytic activity, but the threonine and serine residues are not, although their absence leads to partial inhibition of the catalytic activity (4).…”