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

Abstract

Elucidation of physicochemical mechanisms of enzymatic processes is one of the main tasks of modern biology. High efficiency and selectivity of enzymatic catalysis are mostly ensured by conformational dynamics of enzymes and substrates. Here, we applied a stopped-flow kinetic analysis based on fluorescent spectroscopy to investigate mechanisms of conformational transformations during the removal of alkylated bases from DNA by ALKBH2, a human homolog of Escherichia coli AlkB dioxygenase. This enzyme protects genomic DNA against various alkyl lesions through a sophisticated catalytic mechanism supported by a cofactor (Fe(II)), a cosubstrate (2-oxoglutarate), and O2. We present here a comparative study of conformational dynamics in complexes of the ALKBH2 protein with double-stranded DNA substrates containing N1-methyladenine, N3-methylcytosine, or 1,N6-ethenoadenine. By means of fluorescent labels of different types, simultaneous detection of conformational transitions in the protein globule and DNA substrate molecule was performed. Fitting of the kinetic curves by a nonlinear-regression method yielded a molecular mechanism and rate constants of its individual steps. The results shed light on overall conformational dynamics of ALKBH2 and damaged DNA during the catalytic cycle.

Individual Contributions of Amido Acid Residues Tyr122, Ile168, and Asp173 to the Activity and Substrate Specificity of Human DNA Dioxygenase ABH2

“…Dissociation constants were in a narrow range of 2.1-2.7 µM for the binding to the m 1 A-containing DNA and 2.5-4.2 µM for the binding to the m 3 C-containing DNA (Figure 8). It should be mentioned that these values are in good agreement with data obtained previously in an equilibrium fluorescent titration assay [41].…”

“…It has also been shown that Glu175 and Phe124 contribute to nucleotide-base-specific selection and stabilization in the active site for repair. To elucidate the mechanism of the substrate specificity of the ABH2 enzyme, analyses of conformational changes occurring in the enzyme-substrate complex during the catalytic cycle have been performed by quantum mechanical/molecular mechanical (QM/MM) modeling [39,40] and by the stopped-flow fluorescence spectroscopy technique [41]. The results indicate that the conformational flexibility of protein domains and of the DNA substrate is important for their effective interaction.…”

The Role of Key Amino Acids of the Human Fe(II)/2OG-Dependent Dioxygenase ALKBH3 in Structural Dynamics and Repair Activity toward Methylated DNA

“…This analysis yielded the observed rate constants for demethylation, k obs , which were found to be 0.70 ± 0.08 min −1 and 0.17 ± 0.02 min −1 for the wt and Y143F, respectively. Previously, under identical conditions, the k obs values for another human dioxygenase, ALKBH2, were determined to be 0.22 min −1 for a similar m3C-containing substrate [7]. This difference in rate constants suggests that ALKBH3 exhibits a higher specificity for m3C damage compared to its homologue ALKBH2.…”

“…For example, NMR, time-resolved fluorescence spectroscopy, and circular dichroism (CD) have demonstrated a dynamic behavior of AlkB conformation that accompanies the binding of cofactors and a DNA substrate [21,22]. Using a stopped-flow (SF) approach combined with fluorescence spectroscopy we previously demonstrated how the conformational dynamics of AlkB and ALKBH2 proteins induce and follow the recognition and processing of the alkylated substrate [7,23]. The present work was performed by using biochemical and biophysical approaches.…”

“…ALKBH3 and AlkB have more in common regarding the substrate specificity than do ALKBH3 and ALKBH2, and they can oxidize the methyl group of N1-methyladenine (m1A), N3-methylcytosine (m3C), and N1-methylguanine (m1G) predominantly in single-stranded (ss) DNA or RNA. In contrast to ALKBH2, ALKBH3 repairs m3C more effectively than m1A, whereas AlkB acts equally well against these lesions [6,7]. Although the biological function of ALKBH3 is not completely understood, some data indicate its demethylation activity on ssDNA regions arising during replication or transcription, as well as on m1A-containing mRNA in vivo [8,9].…”