PDBsum entry 3khb

Oxidoreductase

PDB id: 3khb

Contents

Protein chains

203 a.a. *

Ligands

AKG ×2

Metals

_CO ×2

Waters ×30

* Residue conservation analysis

PDB id:

3khb

Name:

Oxidoreductase

Title:

Crystal structure of escherichia coli alkb with co(ii) and 2-og

Structure:

Alpha-ketoglutarate-dependent dioxygenase alkb. Chain: a, b. Synonym: alkylated DNA repair protein alkb. Engineered: yes

Source:

Escherichia coli k-12. Organism_taxid: 83333. Strain: k12. Gene: aidd, alkb, b2212, jw2200. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.90Å R-factor: 0.234 R-free: 0.307

Authors:

T.Hollis,P.J.Holland

Key ref:

P.J.Holland and T.Hollis (2010). Structural and mutational analysis of Escherichia coli AlkB provides insight into substrate specificity and DNA damage searching. Plos One, 5, e8680. PubMed id: 20084272

Date:

30-Oct-09 Release date: 12-Jan-10

Protein chains

P05050  (ALKB_ECOLI) -  Alpha-ketoglutarate-dependent dioxygenase AlkB from Escherichia coli (strain K12)

Seq: 216 a.a.

Struc: 203 a.a.

Enzyme reactions

• Enzyme class: E.C.1.14.11.33 - Dna oxidative demethylase.
• Reaction: a methylated nucleobase within DNA + 2-oxoglutarate + O2 = a nucleobase within DNA + formaldehyde + succinate + CO2

methylated nucleobase within DNA (Bound ligand (Het Group name = AKG) corresponds exactly) + 2-oxoglutarate + O2 = nucleobase within DNA + formaldehyde + succinate + CO2

• Cofactor: Fe cation

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

Plos One 5:e8680 (2010)

PubMed id: 20084272

Structural and mutational analysis of Escherichia coli AlkB provides insight into substrate specificity and DNA damage searching.

P.J.Holland, T.Hollis.

ABSTRACT

BACKGROUND: In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG) iron(II) dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions, but it also repairs 1-methylguanine (1-meG) and 3-methylthymine (3-meT) at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. METHODOLOGY/PRINCIPAL FINDINGS: We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. CONCLUSIONS/SIGNIFICANCE: A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a "searching" mode and "repair" mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.