2',3'-cyclic-nucleotide 3'-phosphodiesterase

 

During the tRNA splicing reaction in eukaryotes, an ADP ribose cyclic phosphate is formed. Hydrolysis of this to regenerate the monoester ADP ribose-1-phosphate is therefore a necessary part of the overall process. The enzyme cyclic phosphodiesterase is able to catalyse this reaction in the organism Arabidopsis thaliania. It displays a unique fold, but has some sequence identity with the equivalent enzymes in both wheat and zebrafish, specifically in the presence of the sequence motif H-X-T-S-X-H in the active site. Mechanistic and active site features in this family of enzymes are also found to be similar to other RNA processing proteins such as RNAase A.

 

Reference Protein and Structure

Sequence
O04147 UniProt (3.1.4.-) IPR012386 (Sequence Homologues) (PDB Homologues)
Biological species
Arabidopsis thaliana (Thale cress) Uniprot
PDB
1jh6 - Semi-reduced Cyclic Nucleotide Phosphodiesterase from Arabidopsis thaliana (1.8 Å) PDBe PDBsum 1jh6
Catalytic CATH Domains
3.90.1140.10 CATHdb (see all for 1jh6)
Click To Show Structure

Enzyme Reaction (EC:3.1.4.37)

2',3'-cyclic nucleotide(1-)
CHEBI:66954ChEBI
+
water
CHEBI:15377ChEBI
→
ribonucleoside 2'-monophosphate(2-)
CHEBI:78552ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: 2',3'-cyclic AMP phosphodiesterase, 2',3'-cyclic nucleoside monophosphate phosphodiesterase, 2',3'-cyclic nucleotide 3'-phosphohydrolase, 2',3'-cyclic nucleotide phosphohydrolase, 2':3'-CNMP-3'-ase, 2':3'-cyclic nucleotide 3'-phosphodiesterase, CNPase, Cyclic 2',3'-nucleotide 3'-phosphodiesterase, Cyclic 2',3'-nucleotide phosphodiesterase, Cyclic-CMP phosphodiesterase,

Enzyme Mechanism

Introduction

The reaction proceeds by nucleophilic attack by a water molecule, activated by His 119, itself primed by Met 117, on the cyclic phosphate substrate. This leads to a pentavalent phosphate intermediate stabilised by Thr 44, Tyr 124 and Ser 121, which collapses following protonation by His 42 of the 2'OH group to leave the product ADP ribose-1-phosphate.

Catalytic Residues Roles

UniProt PDB* (1jh6)
Met117 (main-C) Met117A (main-C) Ensures that His 119 is in the correct protonation state by forming favourable contacts between the carbonyl and the NH group of the Histidine. increase basicity, electrostatic stabiliser
His42 His42A Protonates the 2C of the ribose sugar to allow it to act as a leaving group thus facilitating collapse of the pentavalent phosphate transition state. proton donor
His119 His119A Activates water by deprotonating it so that it can act as a nucleophile and attack the cyclic phosphate. increase nucleophilicity, proton acceptor
Ser121, Tyr124 Ser121A, Tyr124A Stabilises the pentavalent phosphate transition state that forms during the reaction. electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

overall reactant used, proton transfer, bimolecular nucleophilic addition, overall product formed, decyclisation, unimolecular elimination by the conjugate base

References

  1. Hofmann A et al. (2000), EMBO J, 19, 6207-6217. Structure and mechanism of activity of the cyclic phosphodiesterase of Appr>p, a product of the tRNA splicing reaction. DOI:10.1093/emboj/19.22.6207. PMID:11080166.
  2. Myllykoski M et al. (2013), J Mol Biol, 425, 4307-4322. Crystallographic analysis of the reaction cycle of 2',3'-cyclic nucleotide 3'-phosphodiesterase, a unique member of the 2H phosphoesterase family. DOI:10.1016/j.jmb.2013.06.012. PMID:23831225.

Step 1. His119 activates a water molecule for nucleophilic attack on the phosphate this leads to the formation of a penta-covalent intermediate stabilized by Thr 44, Tyr 124 and Ser 121. His119 is primed by the amide group of Met117..

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser121A electrostatic stabiliser
Met117A (main-C) electrostatic stabiliser
Tyr124A electrostatic stabiliser
Thr44A electrostatic stabiliser
His119A increase nucleophilicity
Met117A (main-C) increase basicity
His119A proton acceptor

Chemical Components

overall reactant used, proton transfer, ingold: bimolecular nucleophilic addition

Step 2. The intermediate collapses upon protonation from His42 yielding the product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser121A electrostatic stabiliser
Tyr124A electrostatic stabiliser
Thr44A electrostatic stabiliser
His42A proton donor

Chemical Components

overall product formed, decyclisation, ingold: unimolecular elimination by the conjugate base, proton transfer
Download: Image, Marvin File

Step 1. His119 activates a water molecule for nucleophilic attack on the phosphate this leads to the formation of a penta-covalent intermediate stabilized by Thr 44, Tyr 124 and Ser 121. His119 is primed by the amide group of Met117..

Step 2. The intermediate collapses upon protonation from His42 yielding the product.

Products.

Contributors

Peter Sarkies, Gemma L. Holliday, James Willey