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

• Published in: RNA (Cambridge) 2014-11, Vol.20 (11), p.1697-1705
• Main Authors: Remus, Barbara S, Jacewicz, Agata, Shuman, Stewart
• Format: Article
• Language: English
• Subjects: Adenosine Monophosphate - metabolism; Alanine - metabolism; Amino Acid Motifs; Catalytic Domain; Crystallography, X-Ray; Escherichia coli; Escherichia coli - enzymology; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Models, Molecular; Mutagenesis; Nucleotidases - chemistry; Nucleotidases - metabolism; Phylogeny; Protein Conformation; Protein Structure, Tertiary; Sequence Homology, Amino Acid
• ISSN: 1355-8382; 1469-9001
• DOI: 10.1261/rna.046797.114

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.