Ribonuclease HI

 

Members of the RNase H family hydrolyze the P-O3' bond of the RNA strand of an RNA-DNA hybrid duplex in the presence of divalent cations such as Mg(II) and Mn(II). RNase HI is found in Escherichia coli and is structurally homologous to the RNase H domain of HIV-1 reverse transcriptase, a target for anti-HIV therapy.

Two alternative mechanisms have been proposed for this enzyme. One is a two-metal-ion mechanism where one of the two metal ions activates the attacking hydroxide ion and the other is a general acid-base mechanism where an amino acid residue fulfils this role. Computational studies seem to favour the two metal ion proposal, however NMR and kinetic studies suggest only one metal binds to this protein and that the protein is inactivated by subsequent metal binding events.

 

Reference Protein and Structure

Sequence
P0A7Y4 UniProt (3.1.26.4) IPR022892 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1rdd - CRYSTAL STRUCTURE OF ESCHERICHIA COLI RNASE HI IN COMPLEX WITH MG2+ AT 2.8 ANGSTROMS RESOLUTION: PROOF FOR A SINGLE MG2+ SITE (2.8 Å) PDBe PDBsum 1rdd
Catalytic CATH Domains
3.30.420.10 CATHdb (see all for 1rdd)
Cofactors
Magnesium(2+) (1), Water (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:3.1.26.4)

messenger RNA
CHEBI:33699ChEBI
+
water
CHEBI:15377ChEBI
→
3'-end ribonucleotide(1-) residue
CHEBI:74896ChEBI
+
5'-end ribonucleotide(1-) residue
CHEBI:137923ChEBI
Alternative enzyme names: RNA*DNA hybrid ribonucleotidohydrolase, RNase H, Endoribonuclease H (calf thymus), Hybrid nuclease, Hybrid ribonuclease, Hybridase, Hybridase (ribonuclease H), Endoribonuclease H, Calf thymus ribonuclease H,

Enzyme Mechanism

Introduction

The basic mechanism is SN2-like inline displacement of the 3' end of the scissile phosphate group by a water nucleophile in a single step, passing through a pentavalent transition state. The water nucleophile is held and activated by His 124, which deprotonates the water molecule, enabling attack as a hydroxide ion. His 124 itself is made more basic by hydrogen bonding to the substrate phosphate group 3' to the scissile phosphodiester bond. The 3' hydroxyl leaving group is activated as a leaving group by protonation by a water molecule; the water molecule itself is acidified by the Mg(II) cation and the 2' hydroxyl of the substrate ribose 5' to the scissile phosphodiester bond. The residues Asp 134 and Glu 48 are important for orienting the water molecules.

Catalytic Residues Roles

UniProt PDB* (1rdd)
Glu48 Glu48A Glu48 anchors the water molecule that acts as a general acid and helps activate it. increase basicity, increase acidity
Asp134 Asp134A Asp134 holds the nucleophilic water molecule in place and helps activate it. increase acidity
His124 His124A Activates the water nucleophile by deprotonation. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Asp10, Gly11 (main-C), Asp70 Asp10A, Gly11A (main-C), Asp70A Forms part of the magnesium binding site. metal ligand
*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

proton transfer, bimolecular nucleophilic substitution, overall reactant used, overall product formed, hydrolysis, native state of enzyme regenerated, inferred reaction step

References

  1. Haruki M et al. (2000), Biochemistry, 39, 13939-13944. Catalysis by Escherichia coli ribonuclease HI is facilitated by a phosphate group of the substrate. DOI:10.1021/bi001469+. PMID:11076536.
  2. Haruki M et al. (1994), Eur J Biochem, 220, 623-631. Investigating the role of conserved residue Asp134 in Escherichia coli ribonuclease HI by site-directed random mutagenesis. DOI:10.1111/j.1432-1033.1994.tb18664.x. PMID:8125123.
  3. Katayanagi K et al. (1993), Proteins, 17, 337-346. Crystal structure ofEscherichia coli RNase HI in complex with Mg2+ at 2.8 Å resolution: Proof for a single Mg2+-binding site. DOI:10.1002/prot.340170402. PMID:8108376.

Step 1. His124 deprotonates water, which attacks the phosphate of RNA in a nucleophilic substitution that results in the cleavage of the phosphate bond, the 5' end of the RNA molecule reprotonates from magnesium activated water. Reaction proceeds through a pentavalent transition state. The pro-RP-oxygen of the phosphate group 3' to the scissile phosphodiester bond contributes to orient His124 to the best position for catalytic function through the formation of a hydrogen bond [PMID:11076536].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His124A hydrogen bond donor, hydrogen bond acceptor
Asp10A metal ligand
Gly11A (main-C) metal ligand
Asp70A metal ligand
Glu48A increase acidity
Asp134A increase acidity
His124A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, overall reactant used, overall product formed, hydrolysis

Step 2. Water deprotonates His124, regenerating the enzyme active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His124A hydrogen bond donor
Asp10A metal ligand
Gly11A (main-C) metal ligand
Asp70A metal ligand
Glu48A increase basicity
His124A proton donor

Chemical Components

proton transfer, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. His124 deprotonates water, which attacks the phosphate of RNA in a nucleophilic substitution that results in the cleavage of the phosphate bond, the 5' end of the RNA molecule reprotonates from magnesium activated water. Reaction proceeds through a pentavalent transition state. The pro-RP-oxygen of the phosphate group 3' to the scissile phosphodiester bond contributes to orient His124 to the best position for catalytic function through the formation of a hydrogen bond [PMID:11076536].

Step 2. Water deprotonates His124, regenerating the enzyme active site.

Products.

Introduction

This proposal represents the two metal mechanism. Here the water molecules are activated by Mg(II). Computational studies propose that the reaction proceeds in two steps: (1) catalysed primarily by magnesium ion A and its ligands, a water molecule attacks the scissile phosphate. The transient phosphorane formed as a result of this nucleophilic attack decays by breaking the bond between the phosphate and the ribose oxygen. In the resulting intermediate, the dissociated but unprotonated leaving group forms an alkoxide coordinated to magnesium ion B. (2) The reaction is completed by protonation of the leaving group, with a neutral Asp132 as a likely proton donor.

Catalytic Residues Roles

UniProt PDB* (1rdd)
Asp10 Asp10A Acts as a bridging ligand between the two Mg(II) ions. metal ligand
Asp134 Asp134A Forms part of the first magnesium binding site. Also thought to act as a general acid/base. It has been inferred that this residue starts in a negatively charged state and abstracts a proton from the nucleophilic water molecule, before donating the proton back to the final product. metal ligand, proton acceptor, proton donor
Glu48, His124 Glu48A, His124A It is unclear what the function of these residues is in this proposal. unknown
Gly11 (main-C), Asp70 Gly11A (main-C), Asp70A Forms part of the second magnesium binding site. metal ligand
*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

proton transfer, inferred reaction step, overall reactant used, overall product formed, hydrolysis, bimolecular nucleophilic substitution, native state of enzyme regenerated

References

  1. Nowotny M et al. (2005), Cell, 121, 1005-1016. Crystal Structures of RNase H Bound to an RNA/DNA Hybrid: Substrate Specificity and Metal-Dependent Catalysis. DOI:10.1016/j.cell.2005.04.024. PMID:15989951.
  2. Rosta E et al. (2011), J Am Chem Soc, 133, 8934-8941. Catalytic mechanism of RNA backbone cleavage by ribonuclease H from quantum mechanics/molecular mechanics simulations. DOI:10.1021/ja200173a. PMID:21539371.
  3. De Vivo M et al. (2008), J Am Chem Soc, 130, 10955-10962. Phosphodiester cleavage in ribonuclease H occurs via an associative two-metal-aided catalytic mechanism. DOI:10.1021/ja8005786. PMID:18662000.

Step 1. Asp134 abstracts a proton from the metal bound nucleophilic water.

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Catalytic Residues Roles

Residue Roles
Asp10A metal ligand
Gly11A (main-C) metal ligand
Asp70A metal ligand
Asp134A metal ligand
Glu48A unknown
His124A unknown
Asp134A proton acceptor

Chemical Components

proton transfer, inferred reaction step, overall reactant used

Step 2. The catalytic water is activated by its position in the Mg(II) coordination sphere. The hydroxide attacks the phosphate of RNA in a nucleophilic substitution that results in the cleavage of the phosphate bond, the 5' end of the RNA molecule becomes part of the second Mg(II) coordination sphere. The reaction likely proceeds through a pentavalent transition state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu48A unknown
His124A unknown
Asp10A metal ligand
Gly11A (main-C) metal ligand
Asp70A metal ligand
Asp134A metal ligand

Chemical Components

overall reactant used, overall product formed, hydrolysis, ingold: bimolecular nucleophilic substitution

Step 3. The alkoxide group abstracts a proton from Asp134 to form the second product of the reaction.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu48A unknown
His124A unknown
Asp10A metal ligand
Gly11A (main-C) metal ligand
Asp70A metal ligand
Asp134A metal ligand
Asp134A proton donor

Chemical Components

proton transfer, native state of enzyme regenerated, overall product formed
Download: Image, Marvin File

Step 1. Asp134 abstracts a proton from the metal bound nucleophilic water.

Step 2. The catalytic water is activated by its position in the Mg(II) coordination sphere. The hydroxide attacks the phosphate of RNA in a nucleophilic substitution that results in the cleavage of the phosphate bond, the 5' end of the RNA molecule becomes part of the second Mg(II) coordination sphere. The reaction likely proceeds through a pentavalent transition state.

Step 3. The alkoxide group abstracts a proton from Asp134 to form the second product of the reaction.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Jonathan T. W. Ng