PDBsum entry 2g8h

Hydrolase/RNA/DNA

PDB id: 2g8h

Contents

Protein chain

136 a.a. *

DNA/RNA

Ligands

VO4-VO4-VO4-VO4-VO4-VO4

Metals

_MG ×2

Waters ×235

* Residue conservation analysis

PDB id:

2g8h

Name:

Hydrolase/RNA/DNA

Title:

B. Halodurans rnase h catalytic domain d192n mutant in complex with mg2+ and RNA/DNA hybrid (non-p nick at the active site)

Structure:

5'-r( Up Cp Gp Ap Cp A)-3'. Chain: b. Engineered: yes. 5'-d( Ap Tp Gp Tp Cp G)-3'. Chain: c. Engineered: yes. Ribonuclease h. Chain: a. Fragment: bh-rnase hc.

Source:

Synthetic: yes. Bacillus halodurans. Organism_taxid: 86665. Gene: rnha. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Trimer (from PQS)

Resolution:

1.85Å R-factor: 0.206 R-free: 0.254

Authors:

M.Nowotny,W.Yang

Key ref:

M.Nowotny and W.Yang (2006). Stepwise analyses of metal ions in RNase H catalysis from substrate destabilization to product release. EMBO J, 25, 1924-1933. PubMed id: 16601679 DOI: 10.1038/sj.emboj.7601076

Date:

02-Mar-06 Release date: 25-Apr-06

Protein chain

Q9KEI9  (RNH1_BACHD) -  Ribonuclease H from Halalkalibacterium halodurans (strain ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125)

Seq: 196 a.a.

Struc: 136 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DNA/RNA chains

U-C-G-A-C-A 6 bases

A-T-G-T-C-G 6 bases

Enzyme reactions

• Enzyme class: E.C.3.1.26.4 - ribonuclease H.
• Reaction: Endonucleolytic cleavage to 5'-phosphomonoester.

DOI no: 10.1038/sj.emboj.7601076

EMBO J 25:1924-1933 (2006)

PubMed id: 16601679

Stepwise analyses of metal ions in RNase H catalysis from substrate destabilization to product release.

M.Nowotny, W.Yang.

ABSTRACT

In two-metal catalysis, metal ion A has been proposed to activate the nucleophile and metal ion B to stabilize the transition state. We recently reported crystal structures of RNase H-RNA/DNA substrate complexes obtained at 1.5-2.2 Angstroms. We have now determined and report here structures of reaction intermediate and product complexes of RNase H at 1.65-1.85 Angstroms. The movement of the two metal ions suggests how they may facilitate RNA hydrolysis during the catalytic process. Firstly, metal ion A may assist nucleophilic attack by moving towards metal ion B and bringing the nucleophile close to the scissile phosphate. Secondly, metal ion B transforms from an irregular coordination in the substrate complex to a more regular geometry in the product complex. The exquisite sensitivity of Mg(2+) to the coordination environment likely destabilizes the enzyme-substrate complex and reduces the energy barrier to form product. Lastly, product release probably requires dissociation of metal ion A, which is inhibited by either high concentrations of divalent cations or mutation of an assisting protein residue.

Selected figure(s)

Figure 5.
Figure 5 Structure of the E188A Bh-RNase HC product complex. (A) Stereo view of the active site superimposed with the 2F[o]-F[c] map contoured at 0.9 . The 5'-phosphate is shown as red and yellow ball-and-sticks, Mg^2+ ions as purple spheres labeled with 'A' and 'B', and water molecules as small red spheres. (B, C) Side-by-side comparison of metal ion coordination in the substrate and product complexes. The substrate complex (D192N, PDB: 1ZBL) is shown in green with oxygen atoms in red and nitrogen atoms in blue, and the product complex (E188A) is shown in orange. (D) Stereo view of the superposition of substrate and product complexes.

Figure 6.
Figure 6 Schematic representation of the reaction steps proposed for RNase H. The substrate RNA is shown in pink and products in purple. Coordination of metal ions is highlighted in dark blue, and scissile phosphate in red. Selected hydrogen bonds are shown as blue lines. Black circles represent water molecules. The distance between the two metal ions is indicated in the enzyme–substrate, enzyme–intermediate and enzyme–product complexes.

The above figures are reprinted by permission from Macmillan Publishers Ltd: EMBO J (2006, 25, 1924-1933) copyright 2006.

Figures were selected by an automated process.