Ribonuclease T1

 

Ribonuclease T1 is an extracellular enzyme from Aspergillus oryzae that catalyses the hydrolysis of RNA at guanylyl residues yielding new guanosine 3'-phosphate and 5'-OH ends. It enhances the rate of GpC cleavage by about 10^11-fold. This stable, low molecular weight, monomeric endonuclease is the leading representative of a family of related fungal/bacterial proteins that share sequence and three-dimensional structural similarities.

 

Reference Protein and Structure

Sequence
P00651 UniProt (4.6.1.24) IPR000026 (Sequence Homologues) (PDB Homologues)
Biological species
Aspergillus oryzae RIB40 (Fungus) Uniprot
PDB
1b2m - THREE-DIMENSIONAL STRUCTURE OF RIBONULCEASE T1 COMPLEXED WITH AN ISOSTERIC PHOSPHONATE ANALOGUE OF GPU: ALTERNATE SUBSTRATE BINDING MODES AND CATALYSIS. (2.0 Å) PDBe PDBsum 1b2m
Catalytic CATH Domains
3.10.450.30 CATHdb (see all for 1b2m)
Click To Show Structure

Enzyme Reaction (EC:3.1.27.3)

hydron
CHEBI:15378ChEBI
+
water
CHEBI:15377ChEBI
+
guanylyl-(3'->5')-cytidine(1-)
CHEBI:134204ChEBI
→
cytidine
CHEBI:17562ChEBI
+
guanosine 3'-monophosphate
CHEBI:28072ChEBI
Alternative enzyme names: Aspergillus oryzae ribonuclease, RNase F(1), RNase G, RNase N(1), RNase N(2), RNase Sa, RNase T(1), Binase, Guanyl-specific RNase, Guanyloribonuclease, Ribonuclease C2, Ribonuclease Ch, Ribonuclease F(1), Ribonuclease N(1), Ribonuclease N(3), Ribonuclease PP1, Ribonuclease SA, Ribonuclease U(1), Ribonuclease guaninenucleotido-2'-transferase (cyclizing), RNase N1, Ribonuclease T1, RNase N2,

Enzyme Mechanism

Introduction

The reaction occurs in a two-step process with cleavage of the RNA chain by transesterification of a 5'-phosphoester bond to form a guanosine 2',3'-cyclic phosphate terminus in the first step, followed by hydrolysis of the cyclic phosphate to form a guanosine 3'-phosphate in a second, independent step.

Catalytic Residues Roles

UniProt PDB* (1b2m)
His118 His92A(D) General acid, donates proton to 5' leaving group. proton shuttle (general acid/base)
Phe126, Arg103, Tyr64 Phe100A(D), Arg77A(D), Tyr38A(D) Stabilisation of trigonal bipyramidal phosphorane transition state. electrostatic stabiliser
His66 His40A(D) Role is not entirely clear, may be a general base. proton shuttle (general acid/base)
Glu84 Glu58A(D) General base. proton shuttle (general acid/base)
*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

References

  1. Loverix S et al. (2000), Chem Biol, 7, 651-658. Mechanism of RNase T1: concerted triester-like phosphoryl transfer via a catalytic three-centered hydrogen bond. DOI:10.1016/s1074-5521(00)00005-3. PMID:11048955.
  2. Bombarda E et al. (2010), J Phys Chem B, 114, 1994-2003. pH-Dependent pKaValues in Proteins—A Theoretical Analysis of Protonation Energies with Practical Consequences for Enzymatic Reactions. DOI:10.1021/jp908926w. PMID:20088566.
  3. Arni RK et al. (1999), Biochemistry, 38, 2452-2461. Three-Dimensional Structure of Ribonuclease T1Complexed with an Isosteric Phosphonate Substrate Analogue of GpU:  Alternate Substrate Binding Modes and Catalysis†,‡. DOI:10.1021/bi982612q. PMID:10029539.

Step 1.

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Catalytic Residues Roles

Residue Roles
Tyr38A(D) electrostatic stabiliser
Glu58A(D) proton shuttle (general acid/base)
Arg77A(D) electrostatic stabiliser
His92A(D) proton shuttle (general acid/base)
Phe100A(D) electrostatic stabiliser
His40A(D) proton shuttle (general acid/base)

Chemical Components

Step 1.

Contributors

Alex Gutteridge, Craig Porter, Gemma L. Holliday