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  • Title: Preparation and characterization of the native iron(II)-containing DNA repair AlkB protein directly from Escherichia coli.
    Author: Mishina Y, Chen LX, He C.
    Journal: J Am Chem Soc; 2004 Dec 29; 126(51):16930-6. PubMed ID: 15612731.
    Abstract:
    The Escherichia coli AlkB protein was recently found to repair cytotoxic DNA lesions 1-methyladenine and 3-methylcytosine by using a novel iron-catalyzed oxidative demethylation mechanism. This protein belongs to a family of 2-ketoglutarate-Fe(II)-dependent dioxygenase proteins that utilize iron and 2-ketoglutarate to activate dioxygen for oxidation reactions. We report here the overexpression and isolation of the native Fe(II)-AlkB with a bound cofactor, 2-ketoglutarate, directly from E. coli. UV-vis measurements showed an absorption peak at 560 nm, which is characteristic of a bidentate 2-ketoglutarate bound to an iron(II) ion. Addition of excess amounts of single-stranded DNA to this isolated Fe(II)-AlkB protein caused a 9 nm shift of the 560 nm band to a higher energy, indicating a DNA-binding-induced geometry change of the active site. X-ray absorption spectra of the active site iron(II) in AlkB suggest a five-coordinate iron(II) center in the protein itself and a centrosymmetric six-coordinate iron(II) site upon addition of single-stranded DNA. This geometry change may play important roles in the DNA damage-searching and damage-repair functions of AlkB. These results provide direct evidence for DNA binding to AlkB which modulates the active site iron(II) geometry. The isolation of the native Fe(II)-AlkB also allows for further investigation of the iron(II) center and detailed mechanistic studies of the dioxygen-activation and damage-repair reactions performed by AlkB.
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