Publications

• Characterization of a heat-stable enzyme possessing GTP-dependent RNA ligase activity from a hyperthermophilic archaeon, Pyrococcus furiosus.

  Kanai A., Sato A., Fukuda Y., Okada K., Matsuda T., Sakamoto T., Muto Y., Yokoyama S., Kawai G., Tomita M.

  Using an expression protein library of a hyperthermophilic archaeon, Pyrococcus furiosus, we identified a gene (PF0027) that encodes a protein with heat-stable cyclic nucleotide phosphodiesterase (CPDase) activity. The PF0027 gene encoded a 21-kDa protein and an amino acid sequence that showed app ... >> More

  Using an expression protein library of a hyperthermophilic archaeon, Pyrococcus furiosus, we identified a gene (PF0027) that encodes a protein with heat-stable cyclic nucleotide phosphodiesterase (CPDase) activity. The PF0027 gene encoded a 21-kDa protein and an amino acid sequence that showed approximately 27% identity to that of the 2'-5' tRNA ligase protein, ligT (20 kDa), from Escherichia coli. We found that the purified PF0027 protein possessed GTP-dependent RNA ligase activity and that synthetic tRNA halves bearing 2',3'-cyclic phosphate and 5'-OH termini were substrates for the ligation reaction in vitro. GTP hydrolysis was not required for the reaction, and GTPgammaS enhanced the tRNA ligation activity of PF0027 protein, suggesting that the ligation step is regulated by a novel mechanism. In comparison to the strong CPDase activity of the PF0027 protein, the RNA ligase activity itself was quite weak, and the ligation product was unstable during in vitro reaction. Finally, we used NMR to determine the solution structure of the PF0027 protein and discuss the implications of our results in understanding the role of the PF0027 protein. << Less

  RNA 15:420-431(2009) [PubMed] [EuropePMC]

• Structure and mechanism of E. coli RNA 2',3'-cyclic phosphodiesterase.

  Remus B.S., Jacewicz A., Shuman S.

  2H (two-histidine) phosphoesterase enzymes are distributed widely in all domains of life and are implicated in diverse RNA and nucleotide transactions, including the transesterification and hydrolysis of cyclic phosphates. Here we report a biochemical and structural characterization of the Escheri ... >> More

  2H (two-histidine) phosphoesterase enzymes are distributed widely in all domains of life and are implicated in diverse RNA and nucleotide transactions, including the transesterification and hydrolysis of cyclic phosphates. Here we report a biochemical and structural characterization of the Escherichia coli 2H protein YapD YadP [corrected], which was identified originally as a reversible transesterifying "nuclease/ligase" at RNA 2',5'-phosphodiesters. We find that YapD YadP [corrected] is an "end healing" cyclic phosphodiesterase (CPDase) enzyme that hydrolyzes an HORNA>p substrate with a 2',3'-cyclic phosphodiester to a HORNAp product with a 2'-phosphomonoester terminus, without concomitant end joining. Thus we rename this enzyme ThpR (two-histidine 2',3'-cyclic phosphodiesterase acting on RNA). The 2.0 Å crystal structure of ThpR in a product complex with 2'-AMP highlights the roles of extended histidine-containing motifs (43)HxTxxF(48) and (125)HxTxxR(130) in the CPDase reaction. His43-Nε makes a hydrogen bond with the ribose O3' leaving group, thereby implicating His43 as a general acid catalyst. His125-Nε coordinates the O1P oxygen of the AMP 2'-phosphate (inferred from geometry to derive from the attacking water nucleophile), pointing to His125 as a general base catalyst. Arg130 makes bidentate contact with the AMP 2'-phosphate, suggesting a role in transition-state stabilization. Consistent with these inferences, changing His43, His125, or Arg130 to alanine effaced the CPDase activity of ThpR. Phe48 makes a π-π stack on the adenine nucleobase. Mutating Phe28 to alanine slowed the CPDase by an order of magnitude. The tertiary structure and extended active site motifs of ThpR are conserved in a subfamily of bacterial and archaeal 2H enzymes. << Less

  RNA 20:1697-1705(2014) [PubMed] [EuropePMC]