Kinetic Studies on the 2-Oxoglutarate/Fe(II)-Dependent Nucleic Acid Modifying Enzymes from the AlkB and TET Families

Abstract

Nucleic acid methylations are important genetic and epigenetic biomarkers. The formation and removal of these markers is related to either methylation or demethylation. In this review, we focus on the demethylation or oxidative modification that is mediated by the 2-oxoglutarate (2-OG)/Fe(II)-dependent AlkB/TET family enzymes. In the catalytic process, most enzymes oxidize 2-OG to succinate, in the meantime oxidizing methyl to hydroxymethyl, leaving formaldehyde and generating demethylated base. The AlkB enzyme from Escherichia coli has nine human homologs (ALKBH1-8 and FTO) and the TET family includes three members, TET1 to 3. Among them, some enzymes have been carefully studied, but for certain enzymes, few studies have been carried out. This review focuses on the kinetic properties of those 2-OG/Fe(II)-dependent enzymes and their alkyl substrates. We also provide some discussions on the future directions of this field.

Keywords: 2-OG-dependent enzyme; ALKBH protein; AlkB; FTO; TET; kinetics.

Figure 1.

Proposed mechanisms of oxidative modifications on representative substrates catalyzed by 2-OG/Fe(II)-dependent dioxygenases. (a) m1A repaired by AlkB, ALKBH2 and 3; (b) m5C oxidized by AlkB, ALKBH2 and 3 and TET; (c) εA repaired by AlkB, ALKBH2 and 3; (d) m6A demethylated by ALKBH5; and (e) m6A demethylated by FTO.

Figure 2.

Proposed detailed mechanism of AlkB/TET family enzymes on monoalkyl substrates (exemplified with m3C). The steps include 2-OG binding, DNA/RNA substrate binding, dioxygen binding and activation, 2-OG oxidation, Fe(IV)=O formation, ferryl flip, H abstraction, OH rebound, product release, water binding, etc. Adapted from Scheme 1 in [20].