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

The activation of molecular oxygen for the highly selective functionalization and repair of DNA and RNA nucleobases is achieved by alpha-ketoglutarate (alpha-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.

Automated summary

The activation of molecular oxygen for selective functionalization and repair of DNA and RNA nucleobases is achieved by alpha-ketoglutarate/iron-dependent dioxygenases. This article focuses on the human homologues AlkBH and TET enzymes, discussing the importance of studying enzyme-substrate interactions and turnover for understanding their mode of action. Various analytical methods have been employed to study changes in the active site and overall enzyme structure, and several methods are available to assess enzyme activity. The article aims to provide a comprehensive synopsis for researchers in the field of epigenetic processes and DNA/RNA repair and modification.