Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.4.1 (phosphodiesterase)
 18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Saccharomyces cerevisiae Dbr1 is a 405-amino acid RNA debranching enzyme that cleaves the 2'-5' phosphodiester bonds of the lariat introns formed during pre-mRNA splicing. Debranching appears to be a rate-limiting step for the turnover of intronic RNA, insofar as the steady-state levels of lariat introns are greatly increased in a Deltadbr1 strain. To gain insight to the requirements for yeast Dbr1 function, we performed a mutational analysis of 28 amino acids that are conserved in Dbr1 homologs from other organisms. We identified 13 residues (His13, Asp40, Arg45, Asp49, Tyr68, Tyr69, Asn85, His86, Glu87, His179, Asp180, His231 and His233) at which alanine substitutions resulted in lariat intron accumulation in vivo. Conservative replacements at these positions were introduced to illuminate structure-activity relationships. Residues important for Dbr1 function include putative counterparts of the amino acids that comprise the active site of the metallophosphoesterase superfamily, exemplified by the DNA phosphodiesterase Mre11. Using natural lariat RNAs and synthetic branched RNAs as substrates, we found that mutation of Asp40, Asn85, His86, His179, His231 or His233 to alanine abolishes or greatly diminishes debranching activity in vitro. Dbr1 sediments as a monomer and requires manganese as the metal cofactor for debranching.
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PMID:Structure-function analysis of yeast RNA debranching enzyme (Dbr1), a manganese-dependent phosphodiesterase. 1627 84

2',5'-branched RNA was recently proposed as a key Ty1 retrotransposition intermediate, for which cleavage by lariat debranching enzyme (Dbr1p) enables reverse transcription to continue synthesizing the complete Ty1 cDNA. Because dbr1 cells can produce substantial Ty1 cDNA despite lacking Dbr1p, the obligatory intermediacy of branched RNA would require that Ty1 reverse transcriptase (RT) can read through the proposed branch site with considerable efficiency. Here we have used deoxyribozyme-synthesized 2',5'-branched RNA corresponding exactly to the proposed Ty1 branch site for a direct test of this read-through ability. Using an in vitro assay that incorporates all components known to be required for Ty1 cDNA synthesis (including the TyA chaperone protein), Ty1 RT can elongate up to the branch site. Strand transfer from the 2'-arm to the 3'-arm of the branch is observed when the Ty1 RT is RNase H+ (i.e., wild-type) but not when the Ty1 RT is RNase H-. When elongating from either the 2'-arm or the 3'-arm, Ty1 RT reads through the branch site with <or=0.3% efficiency. This is at least 60-fold lower than would be necessary to explain in vivo Ty1 cDNA synthesis in dbr1 cells, because others have reported 18% cDNA synthesis relative to wild-type cells. Our finding that Ty1 RT cannot efficiently read through the proposed Ty1 branch site is inconsistent with the hypothesis that branched RNA is an obligatory Ty1 retrotransposition intermediate. This suggests that Dbr1p acts as other than a 2',5'-phosphodiesterase during Ty1 retrotransposition.
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PMID:Ty1 reverse transcriptase does not read through the proposed 2',5'-branched retrotransposition intermediate in vitro. 1765 36

Two RNA fragments linked by means of a 2',5' phosphodiester bridge (2' hydroxyl of one fragment connected to the 5' hydroxyl of the other) constitute a class of nucleic acids known as 2'-5' branched RNAs (bRNAs). In this report we show that bRNA analogues containing 2'-5' phosphoramidate linkages (bN-RNAs) inhibit the lariat debranching enzyme, a 2',5'-phosphodiesterase that has recently been implicated in neurodegenerative diseases associated with aging. bN-RNAs were efficiently generated using automated solid-phase synthesis and suitably protected branchpoint building blocks. Two orthogonally removable groups, namely the 4-monomethoxytrityl (MMTr) group and the fluorenylmethyl-oxycarbonyl (Fmoc) groups, were evaluated as protecting groups of the 2' amino functionality. The 2'-N-Fmoc methodology was found to successfully produce bN-RNAs on solid-phase oligonucleotide synthesis. The synthesized bN-RNAs resisted hydrolysis by the lariat debranching enzyme (Dbr1) and, in addition, were shown to attenuate the Dbr1-mediated hydrolysis of native bRNA.
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PMID:Design, Synthesis, and Properties of Phosphoramidate 2',5'-Linked Branched RNA: Toward the Rational Design of Inhibitors of the RNA Lariat Debranching Enzyme. 2637 68

Saccharomyces cerevisiae Dbr1 is a manganese-dependent RNA debranching enzyme that cleaves the 2'-5' phosphodiester bond of the lariat introns formed during pre-mRNA splicing. Dbr1 is a member of the binuclear metallophosphoesterase enzyme superfamily. We showed previously via alanine scanning that RNA debranching in vivo and in vitro depends on conserved active site residues His13, Asp40, Asn85, His86, His179, His231, and His233. Here, by extending the alanine scan, we added Cys11 to the ensemble of essential active site components. We report that Dbr1 has a vigorous manganese-dependent phosphodiesterase activity with the non-RNA substrate bis-p-nitrophenylphosphate. Whereas RNA debranching requires His86, bis-p-nitrophenylphosphatase activity does not. We interpret these and other structure-activity relations reported here in light of the crystal structures of Entamoeba Dbr1 and other homologous binuclear metallophosphodiesterases. Our results suggest that (i) Dbr1 adheres to the two-metal mechanism of the enzyme superfamily, but is distinguished by its reliance on a Cys11-Xaa-His13 motif to engage one of the catalytic metals instead of the Asp-Xaa-His element typical of other clades within the superfamily; (ii) His86 is a general acid catalyst that protonates the O2' leaving group of the RNA 2'-5' phosphodiester; and (iii) the favorable pKa of p-nitrophenol elides the strict need for a general acid during hydrolysis of bis-p-nitrophenylphosphate. The Dbr1 bis-p-nitrophenylphosphatase activity is well suited for high-throughput screening for inhibitors of debranching.
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PMID:Mechanistic insights into the manganese-dependent phosphodiesterase activity of yeast Dbr1 with bis-p-nitrophenylphosphate and branched RNA substrates. 2776 21