Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Jan 15;449(2):491-6.
doi: 10.1042/BJ20121155.

Investigations on the Oxygen Dependence of a 2-oxoglutarate Histone Demethylase

Affiliations
Free PMC article

Investigations on the Oxygen Dependence of a 2-oxoglutarate Histone Demethylase

Elena M Sánchez-Fernández et al. Biochem J. .
Free PMC article

Abstract

Histone N(ϵ)-methyl lysine demethylases are important in epigenetic regulation. KDM4E (histone lysine demethylase 4E) is a representative member of the large Fe(II)/2-oxoglutarate- dependent family of human histone demethylases. In the present study we report kinetic studies on the reaction of KDM4E with O2. Steady-state assays showed that KDM4E has a graded response to O2 over a physiologically relevant range of O2 concentrations. Pre-steady state assays implied that KDM4E reacts slowly with O2 and that there are variations in the reaction kinetics which are dependent on the methylation status of the substrate. The results demonstrate the potential for histone demethylase activity to be regulated by oxygen availability.

Figures

Figure 1
Figure 1. Proposed mechanism of KDM4E
Proposed mechanism of KDM4E based on the consensus mechanism for the 2OG oxygenases: O2 is the final substrate to bind to the KDM4E:Fe(II):2OG:H3K9 complex (I-III) resulting in generation of a Fe(IV)-oxo species that enables hydroxylation of the unactivated C-H bond (IV-VI). The unstable hydroxymethyl product is released as HCHO (VII), to give the N-demethylated product [5,6].]
Figure 2
Figure 2. Variation of KDM4E activity with O2 availability
MALDI-MS was used to determine conversion at 37 °C of H3K9me3 to both H3K9me2 (black squares) and H3K9me1 (black circles), and separately, conversion of H3K9me2 to H3K9me1 (white circles).
Figure 3
Figure 3. UV-visible Absorption Spectroscopy
Absorption spectra of anaerobic KDM4E:Fe(II):2OG and KDM4E:Fe(II):2OG:H3K9 complexes at room temperature (KDM4E:2OG absorbance subtracted). KDM4E, 0.4 mM; 2OG, 4 mM; H3K9 peptides, 2 mM; Fe(II), 100 μM.
Figure 4
Figure 4. Stopped-flow UV-visible Absorption Spectroscopy
(a) UV-Vis absorbance spectra at 320nm from stopped-flow experiments mixing KDM4E:Fe(II):2OG:H3K9me3 8mer (black) or KDM4E:Fe(II):2OG (white) with O2. (b) UV-Vis absorbance spectra at 320nm from stopped-flow experiments mixing KDM4E:Fe(II):2OG:H3K9me3 8mer (black) or KDM4E:Fe(II):2OG:H3K9me3 25mer (white) with O2. (c) UV-Vis absorbance spectra at 320nm from stopped-flow experiments mixing KDM4E:Fe(II):2OG: H3K9me3 8mer (black) or KDM4E:Fe(II):2OG:H3K9me2 8mer (white) with O2. Concentrations before mixing: KDM4E (1.0 mM), Fe(II) (900 μM), 2OG (10 mM), peptide (5 mM) and O2 (1.9 mM). Reactions were carried out at 5 °C. Spectra were recorded over 0.001-1000 s, corrected for absorbance observed on mixing with anaerobic buffer.
Figure 5
Figure 5. Rapid chemical quench in the presence of H3K9me3 8mer peptide
Rapid chemical quench experiments for H3K9me3 8mer peptide revealed that demethylation occurred at 0.014 s−1 (black) and 2OG decarboxylation to succinate (white) at 0.011 s−1 (shown on a logarithmic scale), as determined by MALDI-MS and LC-MS, respectively. (In both cases data were fitted with the equation f = y0+a*(1-exp(-b*x)), using SigmaPlot). Concentrations before mixing were KDM4E (1.0 mM), Fe(II) (900 μM), 2OG (10 mM), peptide (5 mM) and O2 (1.9 mM). Reactions were carried out at 5 °C.

Similar articles

See all similar articles

Cited by 25 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback