Preparation and characterization of the native iron(II)-containing DNA repair AlkB protein directly from Escherichia coli.
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.
Mishina
Yukiko
Y
Department of Chemistry, The University of Chicago, Chicago, IL 60637, USA.
Chen
Lin X
LX
He
Chuan
C
D001665:Binding Sites; D002151:Calorimetry; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D005296:Ferrous Compounds / Q000737:chemistry / Q000302:isolation & purification; D005583:Fourier Analysis; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D013052:Spectrometry, X-Ray Emission; D013056:Spectrophotometry, Ultraviolet
D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D005296:Ferrous Compounds; D006899:Mixed Function Oxygenases; C466427:AlkB protein, E coli