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

Abstract

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 ;27% identity to that of the 29-59 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 29,39-cyclic phosphate and 59-OH termini were substrates for the ligation reaction in vitro. GTP hydrolysis was not required for the reaction, and GTPgS 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.

Aberrant 3′ oligoadenylation of spliceosomal U6 small nuclear RNA in poikiloderma with neutropenia

“…The invariance of the H-x-S motif within the USB1 protein family, despite low overall amino acid sequence identity (Figure 2A), supports a critical role in catalysis. The closest structural homologs (DALI server) 24 are the 2Ј, 5Ј RNA ligases from Pyrococcus horikoshii (PDB 1VDX), 25 Pyrococcus furiosus (PDB 2FYH), 26 and Thermus thermophilus (PDB 1IUH), 27 the central domain of the mammalian A-kinase anchoring protein AKAP18␦ (PDB 2VFY), 28 and a 1Ј-2Ј cyclic nucleotide 2Ј phosphodiesterase (CNPase) from Arabidopsis thaliana involved in tRNA splicing (PDB 1FSI). 29 The signature motif residues of rat AKAP18␦ and human USB1 lie in the same position ( Figure 2B).…”

C16orf57, a gene mutated in poikiloderma with neutropenia, encodes a putative phosphodiesterase responsible for the U6 snRNA 3′ end modification

“…These searches, however, did not reveal any detectable sequence similarity to any other proteins of known structure or function. Using highly sensitive methods for distant homology detection (Ginalski et al 2004) and fold recognition , we mapped Usb1 and hUSB1 to various 2H phosphodiesterase structures, including that of a cyclic nucleotide phosphodiesterase, 1FSI (Hofmann et al 2000), and a 29-59 RNA ligase, 2FYH (Kanai et al 2009). Interestingly, hUSB1 was previously classified as a 2H phosphodiesterase superfamily member (CG16790-like family of eukaryotic ligT ligases) (Mazumder et al 2002).…”

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

“…Also retrieved in the second group was the NMR structure of Pyrococcus furiosus PF0027, a thermophilic enzyme with vigorous 2 ′ ,3 ′ -cyclic phosphodiesterase activity on a tetranucleotide RNA>p substrate yielding an RNA 2 ′ p product, and comparatively weak activity in joining tRNA halves with 2 ′ ,3 ′ -cyclic phosphate and 5 ′ -OH ends (Kanai et al 2009). Two unusual, and somewhat mysterious, features of the strand joining activity of PF0027 (i.e., ones not shared with the E. coli 2H enzyme) include a dependence on GTP and a capacity to join tRNA halves with RNA 2 ′ p and 5 ′ -OH ends (Kanai et al 2009). Mammalian CNPase, for which multiple structures are available (Myllykoski et al 2013), was retrieved in a distinctly lower tier with respect to its homology with ThpR (Z-score 7.3 and 3.5 Å RMSD at 126 positions).…”