title: Structure and mechanism of E. coli RNA 2',3'-cyclic phosphodiesterase.
abstract: 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.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.
authors.forename: Barbara S
authors.initials: BS
authors.lastname: Remus
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.
authors.forename: Agata
authors.initials: A
authors.lastname: Jacewicz
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA s-shuman@ski.mskcc.org.
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D000409:Alanine / Q000378:metabolism; D020816:Amino Acid Motifs; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D009708:Nucleotidases / Q000737:chemistry* / Q000378:metabolism*; D010802:Phylogeny; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
chemical_list: D029968:Escherichia coli Proteins; D000249:Adenosine Monophosphate; D009708:Nucleotidases; C040470:3'-nucleotidase; D000409:Alanine
keywords: 2H phosphoesterase; 3′ end healing; RNA repair