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

Abstract

The activation of molecular oxygen for the highly selective functionalization and repair of DNA and RNA nucleobases is achieved by α-ketoglutarate (α-KG)/iron-dependent dioxygenases. Of special interest are the human homologues AlkBH of Escherichia coli EcAlkB and ten-eleven translocation (TET) enzymes. These enzymes are involved in demethylation or dealkylation of DNA and RNA, although additional physiological functions are continuously being found. Given their importance, studying enzyme-substrate interactions, turnover and kinetic parameters is pivotal for the understanding of the mode of action of these enzymes. Diverse analytical methods, including X-ray crystallography, UV/Vis absorption, electron paramagnetic resonance (EPR), circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy have been employed to study the changes in the active site and the overall enzyme structure upon substrate, cofactor, and inhibitor addition. Several methods are now available to assess the activity of these enzymes. By discussing limitations and possibilities of these techniques for EcAlkB, AlkBH and TET we aim to give a comprehensive synopsis from a bioinorganic point-of-view, addressing researchers from different disciplines working in the highly interdisciplinary and rapidly evolving field of epigenetic processes and DNA/RNA repair and modification.[a] D. Schmidl, N.

Single-Molecule Counting of FTO in Human Breast Tissues Based on a Rolling Circle Transcription Amplification-Driven Clustered Regularly Interspaced Short Palindromic Repeat─Cas12a

“…Indirect approaches are based on the measurement of demethylation co-products (e.g., the reduced form of nicotinamide-adenine dinucleotide, formaldehyde dehydrogenase, and formaldehyde), 16 but they suffer from false positive/negative due to the auto-oxidization and self-hydroxylation of the co-products in the presence of molecular dioxygen. 17 Mass spectrometry 18 and high-performance liquid chromatography (HPLC) strategies 6,19 on the analysis of the demethylated products (e.g., m6A residues), but they require laborious multiple steps with the limitation of low throughput, poor sensitivity, and incompatibility with complex biological samples. 20 Fluorescent methods can measure the conformational changes of RNA/DNA and are suitable for intracellular imaging, 21,22 but they are limited by poor sensitivity without signal amplifications.…”

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

“…It has been suggested earlier that, in the absence of the specific substrate, the side chain of Leu177 may undergo oxidation by the hydroxyl radical generated by uncoupled 2OG decarboxylation [14]. His191 belongs to the conservative triad coordinating the Fe(II) ion, so its replacement with Ala prevents the enzyme from being catalytically Spectroscopic methods have been widely applied to study the 2OG dioxygenases involved in nucleic acid repair/modification due to their capabilities to capture intermediate species and conformational transitions of the enzyme-substrate complex, as well as to explore ligand binding under near-physiological conditions [20]. 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].…”