Structure and mechanism of E. coli RNA 2',3'-cyclic phosphodiesterase Journal Article
| Authors: | Remus, B. S.; Jacewicz, A.; Shuman, S. |
| Article 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, which was identified originally as a reversible transesterifying "nuclease/ligase" at RNA 2',5'-phosphodiesters. We find that YapD 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 43HxTxxF48 and 125HxTxxR130 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. |
| Keywords: | unclassified drug; nonhuman; protein motif; enzyme activity; wild type; alkaline phosphatase; escherichia coli; cyclic amp; nuclease; crystal structure; hydrogen bond; selenium; alanine; catalysis; rna structure; transfer rna; static electricity; hydrolysis; mutagenesis; pyrococcus horikoshii; histidine; transesterification; bacillus subtilis; nucleophile; adenine; thermus thermophilus; rna repair; affinity chromatography; bacterial enzyme; bacterial rna; ribose; rna ligase; phosphodiesterase; arabidopsis thaliana; colorimetry; adenosine diphosphate ribose; nucleic acid base; 2',3' cyclic phosphodiesterase; article; pyrococcus furiosus; 2h phosphoesterase; 3' end healing; thpr enzyme; yapd protein; hanging drop vapor diffusion method; structure guided mutagenesis |
| Journal Title: | RNA |
| Volume: | 20 |
| Issue: | 11 |
| ISSN: | 1355-8382 |
| Publisher: | Cold Spring Harbor Laboratory Press |
| Date Published: | 2014-11-01 |
| Start Page: | 1697 |
| End Page: | 1705 |
| Language: | English |
| DOI: | 10.1261/rna.046797.114 |
| PROVIDER: | scopus |
| PMCID: | PMC4201822 |
| PUBMED: | 25239919 |
| DOI/URL: | |
| Notes: | Export Date: 1 December 2014 -- Source: Scopus |
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