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Investigating the Mechanism of the FeII/2-Oxoglutarate-Dependent Histone Demethylases by a Combined Biochemical/Biophysical Approach

Final Report Summary - HISTONE DEMETHYLASES (Investigating the Mechanism of the FeII/2-Oxoglutarate-Dependent Histone Demethylases by a Combined Biochemical/Biophysical Approach)

Histone lysine demethylases comprise an important family of epigenetic regulatory enzymes. They catalyse the demethylation of tri-, di- and monomethylated lysine residues of histones, thus contributing to either silencing or activation of chromatin. Most histone lysine demethylases belong to the 2-oxoglutarate (2OG) and ferrous iron dependent dioxygenase superfamily. This family utilises molecular oxygen to catalyse the hydroxylation of substrates, in a process coupled with 2OG decarboxylation. The histone demethylase KDM4E, a member of the human KDM4 (JMJD2) subfamily which acts on tri- and dimethylated H3K9 (histone H3 lysine 9), has the structural features typical of Fe(II)/2OG oxygenases. KDM4E catalysis is proposed to occur according to the consensus mechanism for (most) 2OG oxygenases.
Continuing with our investigations into demethylation mechanism and effects of oxygen levels on KDM4E demethylase activity, stopped-flow UV-vis spectroscopy kinetic experiments, along with rapid chemical quench and NMR spectroscopy experiments were conducted under the same conditions. The research findings obtained in the presence of KDM4E:Fe(II):2OG complex with various tri-, di-, mono- and unmethylated substrates (H3K9) after mixing with oxygen-saturated buffer show a peak at 320 nm. It is possible that the 320 nm species observed corresponds to the proposed Fe(IV)=O (ferryl) intermediate (Figure 1), and decay of this feature reflects the abstraction of hydrogen from the substrate as demonstrated for other similarly studied 2OG dependent oxygenases, such as taurine dioxygenase and prolyl-4-hydroxylase. The results reveal that the rate of reaction of the KDM4E:Fe(II):2OG:substrate complex with O2 is slow as observed for prolyl hydroxylase 2 (PHD2), a hypoxia-inducible factor (HIF) hydroxylase with a proposed O2-sensing role. Interestingly, they also reveal that the rate of formation of apparent intermediates (320 nm-absorbing species) is dependent on the methylation state of the substrate (H3K9 me3/me2/me1/me0) and the potential for histone demethylase activity to be regulated by oxygen availability in a biologically relevant manner. Overall the results support a mechanism for histone demethylases proceeding via sequential 2-oxoglutarate, histone, and finally oxygen binding. This work will be of significant biological interest to the wide range of scientists that are interested in the fields of epigenetics and hypoxia as this is the first kinetic study of the reaction of a histone demethylase with O2.