Characterization of the Saccharomyces cerevisiae cyclic nucleotide phosphodiesterase involved in the metabolism of ADP-ribose 1",2"-cyclic phosphate.
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.
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Friedrich Miescher-Institut, PO Box 2543, CH-4002 Basel, Switzerland.
Filipowicz
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D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; C401161:adenosine diphosphate-ribose 1''-2''cyclic phosphate; D000246:Adenosine Diphosphate Ribose; D010727:Phosphoric Diester Hydrolases