RRNA endonuclease

 

Alpha-sarcin is an extracellular cytotoxic protein produced by the mold Aspergillus giganteus. It is a specific ribonuclease which inhibits protein synthesis by cleaving a specific phosphodiester bond in the highly conserved sarcin-ricin loop of the 28S rRNA. The enzyme is a member of a distinctive family of fungal ribonucleases known as ribotoxins, which are produced by different Aspergillus species. The ribotoxins show high sequence homology to each other and are also related to the RNAse T1 subfamily of microbial ribonucleases.

 

Reference Protein and Structure

Sequence
P00655 UniProt (4.6.1.23) IPR004025 (Sequence Homologues) (PDB Homologues)
Biological species
Aspergillus giganteus (Fungus) Uniprot
PDB
1de3 - SOLUTION STRUCTURE OF THE CYTOTOXIC RIBONUCLEASE ALPHA-SARCIN (solution nmr Å) PDBe PDBsum 1de3
Catalytic CATH Domains
3.10.450.30 CATHdb (see all for 1de3)
Click To Show Structure

Enzyme Reaction (EC:3.1.27.10)

water
CHEBI:15377ChEBI
+
poly[(GA)]
CHEBI:137464ChEBI
→
GMP 3'-end residue
CHEBI:53114ChEBI
+
AMP 5'-end residue
CHEBI:53098ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: Alpha-sarcin,

Enzyme Mechanism

Introduction

Alpha-sarcin is a cyclizing RNAse. Deprotonation of the 2' OH group by Glu 96 allows this group to attack the 3' phosphate. The 5' oxygen of the departing nucleotide is protonated by His 137. Subsequent hydrolysis of the produced 2',3'-cyclic phosphate completes the reaction to give the corresponding 3'-mononucleotide.

Catalytic Residues Roles

UniProt PDB* (1de3)
Glu123 Glu96A Deprotonates the 2' OH group of the substrate, allowing this group to attack the 3' phosphate to form the cyclic intermediate. Subsequently protonates the 2' OH group when it forms the leaving group as the cyclic intermediate is hydrolysed. increase nucleophilicity, promote heterolysis, proton acceptor, proton donor
His77 His50A Modifies the pKa of Glu 96, allowing it to deprotonate the 2' OH group. electrostatic stabiliser
His164 His137A Initially protonates the 5' oxygen of the departing nucleotide during formation of the cyclic intermediate. Subsequently acts as a base to activate water to hydrolyse the cyclic intermediate. increase nucleophilicity, promote heterolysis, proton acceptor, proton donor
*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 product formed, overall reactant used, proton transfer, intramolecular nucleophilic substitution, cyclisation, decyclisation, bimolecular nucleophilic substitution, native state of enzyme regenerated

References

  1. Campos-Olivas R et al. (1996), FEBS Lett, 399, 163-165. Structural basis for the catalytic mechanism and substrate specificity of the ribonuclease α-sarcin. DOI:10.1016/s0014-5793(96)01320-8. PMID:8980143.
  2. Pérez-Cañadillas JM et al. (2000), J Mol Biol, 299, 1061-1073. The highly refined solution structure of the cytotoxic ribonuclease α-sarcin reveals the structural requirements for substrate recognition and ribonucleolytic activity. DOI:10.1006/jmbi.2000.3813. PMID:10843858.
  3. Lacadena J et al. (1999), Proteins, 37, 474-484. Role of histidine‐50, glutamic acid‐96, and histidine‐137 in the ribonucleolytic mechanism of the ribotoxin α‐sarcin. DOI:10.1002/(sici)1097-0134(19991115)37:3<474::aid-prot14>3.3.co;2-e. PMID:10591106.
  4. Lacadena J et al. (1998), FEBS Lett, 424, 46-48. The cytotoxin α-sarcin behaves as a cyclizing ribonuclease. DOI:10.1016/s0014-5793(98)00137-9. PMID:9580156.

Step 1. Glu96 deprotonates the 2' hydroxyl group this promotes its nucleophilic attack on the phosphate group forming a cyclic intermediate. His137 protonates the the 5' hydroxyl leaving group promoting the cleavage.This SN2 reaction proceeds via a penta-coordinate transition state stabilized by His50.

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Catalytic Residues Roles

Residue Roles
His50A electrostatic stabiliser
Glu96A increase nucleophilicity
His137A promote heterolysis
His137A proton donor
Glu96A proton acceptor

Chemical Components

overall product formed, overall reactant used, proton transfer, ingold: intramolecular nucleophilic substitution, cyclisation

Step 2. His137 now acting as a base deprotonates a water molecule allowing it to perform a nucleophillic attack on the phosphate group. This leads to the cleavage of the 2' hydroxyl- phosphate bond upon protonation by Glu96.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His50A electrostatic stabiliser
His137A increase nucleophilicity
Glu96A promote heterolysis
His137A proton acceptor
Glu96A proton donor

Chemical Components

decyclisation, overall product formed, ingold: bimolecular nucleophilic substitution, native state of enzyme regenerated, proton transfer
Download: Image, Marvin File

Step 1. Glu96 deprotonates the 2' hydroxyl group this promotes its nucleophilic attack on the phosphate group forming a cyclic intermediate. His137 protonates the the 5' hydroxyl leaving group promoting the cleavage.This SN2 reaction proceeds via a penta-coordinate transition state stabilized by His50.

Step 2. His137 now acting as a base deprotonates a water molecule allowing it to perform a nucleophillic attack on the phosphate group. This leads to the cleavage of the 2' hydroxyl- phosphate bond upon protonation by Glu96.

Products.

Contributors

James Torrance, Gemma L. Holliday, James Willey