PMID:15612731

Citation	Mishina, Y, Chen, LX and He, C (2004) Preparation and characterization of the native iron(II)-containing DNA repair AlkB protein directly from Escherichia coli. J. Am. Chem. Soc. 126:16930-6
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.
Links	PubMed Online version:10.1021/ja045066z
Keywords	Binding Sites; Calorimetry; DNA Damage; DNA Repair; DNA, Single-Stranded/chemistry; Escherichia coli/chemistry; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/biosynthesis; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/isolation & purification; Ferrous Compounds/chemistry; Ferrous Compounds/isolation & purification; Fourier Analysis; Mixed Function Oxygenases/biosynthesis; Mixed Function Oxygenases/chemistry; Mixed Function Oxygenases/genetics; Mixed Function Oxygenases/isolation & purification; Protein Conformation; Spectrometry, X-Ray Emission; Spectrophotometry, Ultraviolet
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