Mechanistic insights into the manganese-dependent phosphodiesterase activity of yeast Dbr1 with bis-p-nitrophenylphosphate and branched RNA substrates

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