PDBsum entry 1k3w

Hydrolase/DNA

PDB id: 1k3w

Contents

Protein chain

254 a.a. *

DNA/RNA

Ligands

SO4 ×3

Metals

_ZN

Waters ×397

* Residue conservation analysis

PDB id:

1k3w

Name:

Hydrolase/DNA

Title:

Crystal structure of a trapped reaction intermediate of the DNA repair enzyme endonuclease viii with DNA

Structure:

5'-d( Gp Gp Cp Tp Tp Cp Ap Tp Cp Cp Tp Gp G)-3'. Chain: b. Engineered: yes. 5'-d( Cp Cp Ap Gp Gp Ap (Ped)p Gp Ap Ap Gp Cp C)-3'. Chain: c. Engineered: yes. Endonuclease viii. Chain: a. Engineered: yes

Source:

Synthetic: yes. Escherichia coli. Organism_taxid: 562. Gene: nei. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Hexamer (from PQS)

Resolution:

1.42Å R-factor: 0.165 R-free: 0.203

Authors:

G.Golan,D.O.Zharkov,R.Gilboa,A.S.Fernandes,J.H.Kycia,S.E.Gerchman, R.A.Rieger,A.P.Grollman,G.Shoham

Key ref:

D.O.Zharkov et al. (2002). Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate. EMBO J, 21, 789-800. PubMed id: 11847126 DOI: 10.1093/emboj/21.4.789

Date:

04-Oct-01 Release date: 04-Oct-02

Protein chain

P50465  (END8_ECOLI) -  Endonuclease 8 from Escherichia coli (strain K12)

Seq: 263 a.a.

Struc: 254 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

DNA/RNA chains

T-C-A-T-C-C-T-G 8 bases

C-A-G-G-A-PED-G-A-A-G-C 11 bases

Enzyme reactions

• Enzyme class 2: E.C.3.2.2.- - ?????
• Enzyme class 3: E.C.4.2.99.18 - DNA-(apurinic or apyrimidinic site) lyase.
• Reaction: 2'-deoxyribonucleotide-(2'-deoxyribose 5'-phosphate)- 2'-deoxyribonucleotide-DNA = a 3'-end 2'-deoxyribonucleotide-(2,3- dehydro-2,3-deoxyribose 5'-phosphate)-DNA + a 5'-end 5'-phospho- 2'-deoxyribonucleoside-DNA + H⁺

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

DOI no: 10.1093/emboj/21.4.789

EMBO J 21:789-800 (2002)

PubMed id: 11847126

Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate.

D.O.Zharkov, G.Golan, R.Gilboa, A.S.Fernandes, S.E.Gerchman, J.H.Kycia, R.A.Rieger, A.P.Grollman, G.Shoham.

ABSTRACT

Endonuclease VIII (Nei) of Escherichia coli is a DNA repair enzyme that excises oxidized pyrimidines from DNA. Nei shares with formamidopyrimidine-DNA glycosylase (Fpg) sequence homology and a similar mechanism of action: the latter involves removal of the damaged base followed by two sequential beta-elimination steps. However, Nei differs significantly from Fpg in substrate specificity. We determined the structure of Nei covalently crosslinked to a 13mer oligodeoxynucleotide duplex at 1.25 A resolution. The crosslink is derived from a Schiff base intermediate that precedes beta-elimination and is stabilized by reduction with NaBH(4). Nei consists of two domains connected by a hinge region, creating a DNA binding cleft between domains. DNA in the complex is sharply kinked, the deoxyribitol moiety is bound covalently to Pro1 and everted from the duplex into the active site. Amino acids involved in substrate binding and catalysis are identified. Molecular modeling and analysis of amino acid conservation suggest a site for recognition of the damaged base. Based on structural features of the complex and site-directed mutagenesis studies, we propose a catalytic mechanism for Nei.

Selected figure(s)

Figure 4.
Figure 4 Scheme of Nei−DNA interactions. Nucleotides are numbered beginning from Tg^0, positive towards the 5'-end, with superscript in parentheses for the complementary strand. Only the central 9 bp are shown. Hydrogen bonds are shown as arrows pointing towards their respective acceptors. Leu70, Tyr71 and Pro253 form van der Waals contacts with nearby DNA residues.

Figure 8.
Figure 8 Central region of the DNA duplex in the Nei−DNA complex. Protein residues that fill the void in DNA created by Tg eversion are shown as solid (cyan) spheres. DNA is presented as a ball-and-stick model with dotted van der Waals radii; dRbl in red, the remaining DNA in yellow.

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (2002, 21, 789-800) copyright 2002.

Figures were selected by an automated process.