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Open AccessArticle

A Single-Turnover Kinetic Study of DNA Demethylation Catalyzed by Fe(II)/α-Ketoglutarate-Dependent Dioxygenase AlkB

Institute of Chemical Biology and Fundamental Medicine (ICBFM), 8 Lavrentiev Ave., Novosibirsk 630090, Russia
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Academic Editor: Sérgio F. Sousa
Molecules 2019, 24(24), 4576; https://doi.org/10.3390/molecules24244576
Received: 17 October 2019 / Revised: 26 November 2019 / Accepted: 11 December 2019 / Published: 13 December 2019
(This article belongs to the Special Issue Structure and Function of Metalloenzymes)
AlkB is a Fe(II)/α-ketoglutarate-dependent dioxygenase that repairs some alkylated bases of DNA and RNA in Escherichia coli. In the course of catalysis, oxidation of a co-substrate (α-ketoglutarate, αKG) leads to the formation of a highly reactive ‘oxyferryl’ enzyme-bound intermediate, Fe(IV) = O, ensuring hydroxylation of the alkyl nucleobase adducts. Previous studies have revealed that AlkB is a flexible protein and can adopt different conformations during interactions with cofactors and DNA. To assess the conformational dynamics of the enzyme in complex with single- or double-stranded DNA in real-time mode, we employed the stopped-flow fluorescence method. N1-Methyladenine (m1A) introduced into a sequence of 15-mer oligonucleotides was chosen as the specific damage. Single-turnover kinetics were monitored by means of intrinsic fluorescence of the protein’s Trp residues, fluorescent base analogue 2-aminopurine (2aPu), and a dye–quencher pair (FAM/BHQ1). For all the fluorescent labels, the fluorescent traces showed several phases of consistent conformational changes, which were assigned to specific steps of the enzymatic process. These data offer an overall picture of the structural dynamics of AlkB and DNA during their interaction. View Full-Text
Keywords: dioxygenase AlkB; DNA demethylation; conformational dynamics; protein–DNA interactions; stopped-flow method; single-turnover kinetics; fluorescent probes; FRET analysis dioxygenase AlkB; DNA demethylation; conformational dynamics; protein–DNA interactions; stopped-flow method; single-turnover kinetics; fluorescent probes; FRET analysis
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Kanazhevskaya, L.Y.; Alekseeva, I.V.; Fedorova, O.S. A Single-Turnover Kinetic Study of DNA Demethylation Catalyzed by Fe(II)/α-Ketoglutarate-Dependent Dioxygenase AlkB. Molecules 2019, 24, 4576.

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