PDBsum entry 2jc4

Hydrolase

PDB id: 2jc4

Contents

Protein chain

256 a.a. *

Ligands

1PE

ACT

2HP

Metals

_MG

_NA ×2

Waters ×287

* Residue conservation analysis

PDB id:

2jc4

Name:

Hydrolase

Title:

3'-5' exonuclease (nexo) from neisseria meningitidis

Structure:

Exodeoxyribonuclease iii. Chain: a. Synonym: neisseria meningitidis 3'-5' exonuclease-nexo. Engineered: yes

Source:

Neisseria meningitidis. Organism_taxid: 487. Strain: mc58 serogroup b. Atcc: 23251. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.90Å R-factor: 0.152 R-free: 0.202

Authors:

E.P.Carpenter,A.Corbett,H.Thomson,J.Adacha,K.Jensen,J.Bergeron, I.Kasampalidis,R.Exley,M.Winterbotham,C.Tang,G.Baldwin,P.Freemont

Key ref:

E.P.Carpenter et al. (2007). AP endonuclease paralogues with distinct activities in DNA repair and bacterial pathogenesis. EMBO J, 26, 1363-1372. PubMed id: 17318183 DOI: 10.1038/sj.emboj.7601593

Date:

19-Dec-06 Release date: 06-Mar-07

Protein chain

Q9K100  (Q9K100_NEIMB) -  Exodeoxyribonuclease III from Neisseria meningitidis serogroup B (strain ATCC BAA-335 / MC58)

Seq: 256 a.a.

Struc: 256 a.a.

Enzyme reactions

• Enzyme class: E.C.3.1.11.2 - exodeoxyribonuclease Iii.
• Reaction: Degradation of double-stranded DNA. It acts progressively in a 3'- to 5'-direction, releasing nucleoside 5'-phosphates.

DOI no: 10.1038/sj.emboj.7601593

EMBO J 26:1363-1372 (2007)

PubMed id: 17318183

AP endonuclease paralogues with distinct activities in DNA repair and bacterial pathogenesis.

E.P.Carpenter, A.Corbett, H.Thomson, J.Adacha, K.Jensen, J.Bergeron, I.Kasampalidis, R.Exley, M.Winterbotham, C.Tang, G.S.Baldwin, P.Freemont.

ABSTRACT

Oxidative stress is a principal cause of DNA damage, and mechanisms to repair this damage are among the most highly conserved of biological processes. Oxidative stress is also used by phagocytes to attack bacterial pathogens in defence of the host. We have identified and characterised two apurinic/apyrimidinic (AP) endonuclease paralogues in the human pathogen Neisseria meningitidis. The presence of multiple versions of DNA repair enzymes in a single organism is usually thought to reflect redundancy in activities that are essential for cellular viability. We demonstrate here that these two AP endonuclease paralogues have distinct activities in DNA repair: one is a typical Neisserial AP endonuclease (NApe), whereas the other is a specialised 3'-phosphodiesterase Neisserial exonuclease (NExo). The lack of AP endonuclease activity of NExo is shown to be attributable to the presence of a histidine side chain, blocking the abasic ribose-binding site. Both enzymes are necessary for survival of N. meningitidis under oxidative stress and during bloodstream infection. The novel functional pairing of NExo and NApe is widespread among bacteria and appears to have evolved independently on several occasions.

Selected figure(s)

Figure 3.
Figure 3 His^167 is a structural determinant for the lack of AP endonuclease activity in NExo. (A) Comparison of the active site and the abasic ribose-binding pockets in NExo (yellow), NApe (green) and HAP1 (red), with the DNA from the HAP1/15mer DNA complex (pdb: 1dew). The numbering is for NExo, the equivalent residue numbers for other structures are shown in Figure 2B and Supplementary Figure S2. (B) The abasic ribose-binding site of HAP1 with a 15-mer DNA complex is shown with the electrostatic potential surface of the HAP1 protein (blue and red representing positive and negative charge, respectively) and the superimposed NExo protein (yellow), showing the presence of His^167 of NExo in the proposed abasic ribose-binding site. Atoms in the DNA structure are coloured cyan for carbons, red for oxygen, blue for nitrogen and orange for phosphate. Residues Arg^177 and Met^270 were removed from the HAP1 structure to reveal the binding site beneath. (C) The 25U substrate (100 nM) was incubated with NExo, NExo His^167Ser or NExo His^167Gly (500 nM) for the time periods shown before separation by denaturing PAGE. Control lanes show the unreacted 25U and 25AP substrate after treatment with NaOH. (D) The 25AP substrate (100 nM) was incubated with NExo, NExo His^167Ser or NExo His^167Gly (500 nM) for the time periods shown before separation by denaturing PAGE. Control lanes show the unreacted 25AP substrate and the 25AP substrate after treatment with NaOH. Bands corresponding to 9 and 10 base products may arise through progressive exonuclease activity or through direct AP endonuclease activity to give a 10 base product followed by subsequent exonuclease activity to the 9 base product. (E) Quantification of the combined intensity in the 9 and 10 base products in panel D demonstrates that these accumulate only slowly with wild-type NExo (filled circles) but with NExo His167Gly (grey triangles) and His167Ser (open squares), these products accumulate rapidly and to near completion.

Figure 6.
Figure 6 CI of nexo, nape and nexo:nape. Animals received a 1:1 mixture of wild-type and mutant bacteria, and the C.I. was determined from the ratio of wild-type to mutant in the bloodstream 8 h after inoculation. Both nexo and nape were significantly attenuated, and nexo:nape is more markedly attenuated than either of the single mutant strains, exhibiting an additive effect from the combined mutations. Error bars show the s.e.

The above figures are reprinted by permission from Macmillan Publishers Ltd: EMBO J (2007, 26, 1363-1372) copyright 2007.

Figures were selected by an automated process.