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

Abstract

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. N 1 -Methyladenine (m 1 A) 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.

6‐phenylpyrrolocytosine as a fluorescent probe to examine nucleotide flipping catalyzed by a DNA repair protein

“…proteins. [47][48][49][50][51] Moreover, similar two-step binding processes preceding flipping have been reported for proteins with AGT-like functions based on transient kinetic studies. [43,52,53] Our kinetic analysis suggests that this putative conformational change following initial association of AGT with DNA is nearly irreversible such that the dependence on the concentration of AGT is lost in the two slower 1/τ values derived from the multiexponential fit of the time course.…”

“…It is not unexpected that environmental effects on the fluorescent probe would be observed during the binding process because distortions of this type have been reported based on rapid kinetic studies as well as investigations of the crystal structures of DNA in complex with other base‐flipping proteins. [ 47–51 ] Moreover, similar two‐step binding processes preceding flipping have been reported for proteins with AGT‐like functions based on transient kinetic studies. [ 43,52,53 ] Our kinetic analysis suggests that this putative conformational change following initial association of AGT with DNA is nearly irreversible such that the dependence on the concentration of AGT is lost in the two slower 1/τ values derived from the multiexponential fit of the time course.…”

Conformational Dynamics of Human ALKBH2 Dioxygenase in the Course of DNA Repair as Revealed by Stopped-Flow Fluorescence Spectroscopy

“…During the selection of the kinetic scheme, its sequential complication was carried out, and deviations of the theoretical curves from the experimental ones were evaluated. The rate constants for each elementary step were obtained via optimization of the parameters during numerical integration of the system of differential equations [ 25 , 40 ]. As a result, it was determined that the lowest number of steps needed to describe the experimental curves for the demethylation of substrate m1A is five.…”

“…All of the aforementioned features offer great diversity of catalytic-cleft size, conformational mobility, and other functional characteristics of AlkB family dioxygenases. In our recent study, we attempted to probe single-turnover conformational dynamics of EcAlkB in the course of damaged-DNA repair by stopped-flow fluorescent spectroscopy [ 25 ].…”

Spectroscopic and in vitro Investigations of Fe2+/α‐Ketoglutarate‐Dependent Enzymes Involved in Nucleic Acid Repair and Modification

“…were able to propose a kinetic model for the conformational behavior of EcAlkB upon metal and DNA binding, based on the temporal course of the Trp fluorescence signal [58] . This spectroscopic approach has been extended to track the fluorescence of 2‐aminopurine, which had been placed 3′ to the 1mdA lesion to be repaired by EcAlkB in DNA, and to measure Förster Resonance Energy Transfer (FRET) when an oligonucleotide modified with a 5′‐Fluorescein Amidite (FAM) fluorophore and a 3′‐Black Hole Quencher 1 (BHQ1) quencher was employed [59] . This allowed for approaching EcAlkB protein dynamics from multiple angles.…”