Publications

• A highly specific phosphatase that acts on ADP-ribose 1''-phosphate, a metabolite of tRNA splicing in Saccharomyces cerevisiae.

  Shull N.P., Spinelli S.L., Phizicky E.M.

  One molecule of ADP-ribose 1'',2''-cyclic phosphate (Appr>p) is formed during each of the approximately 500 000 tRNA splicing events per Saccharomyces cerevisiae generation. The metabolism of Appr>p remains poorly defined. A cyclic phosphodiesterase (Cpd1p) has been shown to convert Appr>p to ADP- ... >> More

  One molecule of ADP-ribose 1'',2''-cyclic phosphate (Appr>p) is formed during each of the approximately 500 000 tRNA splicing events per Saccharomyces cerevisiae generation. The metabolism of Appr>p remains poorly defined. A cyclic phosphodiesterase (Cpd1p) has been shown to convert Appr>p to ADP-ribose-1''-phosphate (Appr1p). We used a biochemical genomics approach to identify two yeast phosphatases that can convert Appr1p to ADP-ribose: the product of ORF YBR022w (now Poa1p), which is completely unrelated to other known phosphatases; and Hal2p, a known 3'-phosphatase of 5',3'-pAp. Poa1p is highly specific for Appr1p, and thus likely acts on this molecule in vivo. Poa1 has a relatively low K(M) for Appr1p (2.8 microM) and a modest kcat (1.7 min(-1)), but no detectable activity on several other substrates. Furthermore, Poa1p is strongly inhibited by ADP-ribose (K(I), 17 microM), modestly inhibited by other nucleotides containing an ADP-ribose moiety and not inhibited at all by other tested molecules. In contrast, Hal2p is much more active on pAp than on Appr1p, and several other tested molecules were Hal2p substrates or inhibitors. poa1-Delta mutants have no obvious growth defect at different temperatures in rich media, and analysis of yeast extracts suggests that approximately 90% of Appr1p processing activity originates from Poa1p. << Less

  Nucleic Acids Res. 33:650-660(2005) [PubMed] [EuropePMC]

• Structure and mechanism of ADP-ribose-1''-monophosphatase (Appr-1''-pase), a ubiquitous cellular processing enzyme.

  Kumaran D., Eswaramoorthy S., Studier F.W., Swaminathan S.

  Appr-1''-pase, an important and ubiquitous cellular processing enzyme involved in the tRNA splicing pathway, catalyzes the conversion of ADP-ribose-1''monophosphate (Appr-1''-p) to ADP-ribose. The structures of the native enzyme from the yeast and its complex with ADP-ribose were determined to 1.9 ... >> More

  Appr-1''-pase, an important and ubiquitous cellular processing enzyme involved in the tRNA splicing pathway, catalyzes the conversion of ADP-ribose-1''monophosphate (Appr-1''-p) to ADP-ribose. The structures of the native enzyme from the yeast and its complex with ADP-ribose were determined to 1.9 A and 2.05 A, respectively. Analysis of the three-dimensional structure of this protein, selected as a target in a structural genomics project, reveals its putative function and provides clues to the catalytic mechanism. The structure of the 284-amino acid protein shows a two-domain architecture consisting of a three-layer alphabetaalpha sandwich N-terminal domain connected to a small C-terminal helical domain. The structure of Appr-1''-pase in complex with the product, ADP-ribose, reveals an active-site water molecule poised for nucleophilic attack on the terminal phosphate group. Loop-region residues Asn 80, Asp 90, and His 145 may form a catalytic triad. << Less

  Protein Sci. 14:719-726(2005) [PubMed] [EuropePMC]