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JOURNAL ARTICLE
RESEARCH SUPPORT, N.I.H., EXTRAMURAL
Mechanistic insights into the manganese-dependent phosphodiesterase activity of yeast Dbr1 with bis-p-nitrophenylphosphate and branched RNA substrates.
RNA 2016 December
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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