Changes in Protein Dynamics of the DNA Repair Dioxygenase AlkB upon Binding of Fe²⁺ and 2-Oxoglutarate

Abstract

The Escherichia coli DNA repair enzyme AlkB is a 2-oxoglutarate (2OG)-dependent Fe(2+) binding dioxygenase that removes methyl lesions from DNA and RNA. To date, nine human AlkB homologues are known: ABH1 to ABH8 and the obesity-related FTO. Similar to AlkB, these homologues exert their activity on nucleic acids, although for some homologues the biological substrate remains to be identified. 2OG dioxygenases require binding of the cofactors Fe(2+) and 2OG in the active site to form a catalytically competent complex. We present a structural analysis of AlkB using NMR, fluorescence, and CD spectroscopy to show that AlkB is a dynamic protein exhibiting different folding states. In the absence of the cofactors Fe(2+) and 2OG, apoAlkB is a highly dynamic protein. Binding of either Fe(2+) or 2OG alone does not significantly affect the protein dynamics. Formation of a fully folded and catalytically competent holoAlkB complex only occurs when both 2OG and Fe(2+) are bound. These findings provide the first insights into protein folding of 2OG-dependent dioxygenases. A role for protein dynamics in the incorporation of the metal cofactor is discussed.

Protein Dynamics Control the Progression and Efficiency of the Catalytic Reaction Cycle of the Escherichia coli DNA-Repair Enzyme AlkB

“…Notably, the Mn(II)/Suc complex does not release the pentamer substrate more rapidly than the Mn(II)/2OG complex (Fig. 14), which is contrary to previous claims (32,35) but is consistent with the need to sequester the oxyferryl intermediate in the active site after Suc generation.…”

“…Significant ligand-dependent protein conformational changes have been observed in several Fe(II)/ 2OG dioxygenases, including AlkB (32), as well as in some homologous halogenases for which crystal structures have been solved in open or closed conformations depending on 2OG binding (33,34). Changes in resonance frequencies and line shapes in 1 H one-dimensional and 1 H-15 N two-dimensional NMR spectra also suggest that the conformational dynamics of AlkB are ligand-dependent (32,35), and these data have been interpreted to reflect increased backbone dynamics that serve to accelerate ligand release from the enzyme upon oxidation of 2OG to Suc. However, given the slow reaction rate of the oxyferryl intermediate generated in parallel with Suc, such accelerated ligand release would be expected to promote quenching of the oxyferryl intermediate (due to premature release of the primary substrate) and adventitious release of reactive oxygen species.…”

DNA Repair by Reversal of DNA Damage

“…These observations provide direct support of the oxidative-demethylation mechanism. Together with solution observations (Bleijlevens et al 2008(Bleijlevens et al , 2012, a more complete picture of cofactor binding, substrate flipping and processing, and product release is now emerging.…”

Homology modeling, molecular dynamics, and site-directed mutagenesis study of AlkB human homolog 1 (ALKBH1)

“…QM/MM calculations and energy decomposition analysis performed for AlkB revealed a total of 9 residues around its active site that are important for the catalytic activity of the enzyme [14]. AlkB is a dynamic protein and when it has no bound cofactors it is mobile and can have different conformations [15]. …”