Is JmjC oxygenase catalysis limited to demethylation?

Article abstract of DOI:10.1002/anie.201303282

Jobs on the side: Substrate selectivity studies indicate that members of the biomedically important JmjC demethylase family of histone N ^ε-methyllysine demethylases are capable of catalyzing the de-N-alkylation of groups other than N-methyl and

Full text of DOI:10.1002/anie.201303282

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Chemie
DOI: 10.1002/anie.201303282
Demethylases/Hydroxylases
Is JmjC Oxygenase Catalysis Limited to Demethylation?**
Richard J. Hopkinson, Louise J. Walport, Martin Mꢀnzel, Nathan R. Rose, Tristan J. Smart,
Akane Kawamura, Timothy D. W. Claridge, and Christopher J. Schofield*
Histone modifications are of central importance
in the regulation of transcription.[1,2] Whilst his-
tone acetylation is in general transcriptionally
activating, histone lysine methylation can be
activating or inhibitory depending on factors
including the site and type of modification.
Therefore, modulation of histone methylation is
being pursued for the therapeutic regulation of
gene expression.[3–6]
The JmjC family of Ne-methyllysine histone
demethylases are ferrous iron and 2-oxoglutarate
(2OG) oxygenases that are likely present in all
animals.[7–9] Unlike the flavin-dependent lysine-
specific demethylases, the larger JmjC demethy-
lase family accepts all 3 Ne-lysine methylation
states. JmjC catalysis is proposed to proceed by
means of hydroxylation to give a hemiaminal
intermediate, which fragments to give the deme-
thylated product and formaldehyde (Figure 1);
however, to date the hemiaminal has not been
observed.[10,11] Although there have been studies
on the sequence and methylation-state selectiv-
ities of JmjC enzymes,[12–15] currently there are no
reports on whether they can oxidize N-alkyl
groups other than methyl or on their selectivity
for the lysine side chain. Here we report sub-
strate–selectivity studies with representative
human JmjC demethylases, which reveal their
potential to act on N-alkyl groups other than
methyl, and to catalyze hydroxylation of groups
Figure 1. Top: View from a crystal structure of JMJD2A (KDM4A) (PDB ID: 2OQ6,
other than N-methyl.
with Ni substituted for Fe).[11] Bottom: Mechanism of Ne-dealkylation catalyzed by
To investigate the selectivity of JmjC deme-
JmjC demethylases. During demethylation, the hemiaminal intermediate is proposed
to spontaneously fragment to give the demethylated lysine and HCHO. The
thylases with respect to N-alkyl groups other than
methyl, a set of histone H3 fragment peptides
incorporating Ne-alkyl lysines at histone H3
lysine-9 and lysine-36 were synthesized and
structures of lysine analogues used in this work are boxed.
tested for reaction with representative JmjC demethylases
JMJD2E (KDM4E), PHF8, and FBXL11 (KDM2A). These
enzymes were chosen because of their different sequence and
methylation-state selectivities: JMJD2E acts on H3 Lysine-
[*] Dr. R. J. Hopkinson, L. J. Walport, Dr. M. Mꢀnzel, Dr. N. R. Rose,
T. J. Smart, Dr. A. Kawamura, Dr. T. D. W. Claridge,
Prof. C. J. Schofield
Fellowship to M.M.). Assistance with NMR characterization from
Ivanhoe K. H. Leung is gratefully acknowledged.
Supporting information for this article (including details on the
synthesis of lysine analogues and peptides, assay methods, MALDI-
TOF, and NMR analyses) is available on the WWW under http://dx.
doi.org/10.1002/anie.201303282.
Chemistry Research Laboratory, University of Oxford
12 Mansfield Road, Oxford, OX1 3TA (UK)
E-mail: christopher.schofield@chem.ox.ac.uk
[**] We thank the Wellcome Trust, the Biotechnology and Biomedical
Sciences Research Council, and the British Heart Foundation Centre
of Research Excellence Oxford (RE/08/004) for funding this work.
We also thank the People Programme (Marie Curie Actions) of the
European Union’s Seventh Framework Programme (FP7/2007-
2013) under REA grant agreement no. 298603 (Marie Curie IEF
ꢁ 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
KGaA. This is an open access article under the terms of the Creative
Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly
cited.
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Figure 2. JmjC demethylases catalyze dealkylation and hydroxylation reactions. Mass spectra of incubations of a) ART-Lys(Me3)-QTAR-Lys(Me/Et)-
STGGKA + JMJD2E, b) ART-Lys(Me3)-QTAR-Lys(Me/Et)-STGGKA + PHF8, c) PATGGV-Lys(Me/Et)-KPHRY + FBXL11, d) ART-Lys(Me3)-QTAR-
Lys(iPr)-STGGKA + JMJD2E, e) ART-Lys(Me3)-QTAR-Lys(iPr)-STGGKA + PHF8, f) ART-Lys(Me3)-QTAR-Lys(Me/iPr)-STGGKA + JMJD2E,
g) PATGGV-Lys(Me/iPr)-KPHRY + FBXL11, h) ART-Lys(Me3)-QTAR-Orn(Me2)-STGGKA + PHF8, and i) PATGGV-Orn(Me2)-KPHRY + FBXL11.
Spectra of the corresponding substrate peptides are shown in red. MS timecourses for selected reactions: j) ART-Lys(Me3)-QTAR-Lys(Me/Et)-
STGGKA + JMJD2E, k) ART-Lys(Me3)-QTAR-Lys(Me/Et)-STGGKA + PHF8, l) ART-Lys(Me3)-QTAR-Lys(Me/iPr)-STGGKA + JMJD2E.
9(Me3/Me2/Me1),[11] PHF8 acts on H3 Lysine-9(Me2/Me1),[16]
and FBXL11 acts on H3 Lysine-36(Me2/Me1).[17] We began by
synthesizing Ne-diethyllysine (Lys(Et2)) analogues of the Ne-
dimethyllysine substrates for the JmjC demethylases. How-
ever, in no case did we observe reaction under standard
conditions when using MS-based assays. We then prepared
the corresponding dialkylated Ne-methyl-Ne-ethyllysine (Lys-
(Me/Et)) analogues. In contrast to the Lys(Et2) analogues,
clear mass shifts were observed when they were incubated
with each of the enzymes JMJD2E, PHF8, and FBXL11
under appropriate conditions.
occur under standard conditions (Figure 2b). However, no
evidence for significant formation of Ne-monomethyllysine
peptide (Lys(Me1)) was accrued, although the Ne-monoethyl-
lysine peptide (Lys(Et1)) was detected in time course experi-
ments (Figure 2k). Competition experiments with JMJD2E
between the H3 Lysine-9(Me/Et) peptide (15-mer, ART-
Lys(Me3)-QTAR-Lys(Me/Et)-STGGKA) and a closely anal-
ogous dimethyllysine peptide (14-mer, ART-Lys(Me3)-
QTAR-Lys(Me2)-STGGK) provided evidence for the forma-
tion of the monomethyllysine peptide derived from the
dimethyllysine peptide (14-mer), as well as for the mono-
ethyllysine peptide and both 14-mer and 15-mer unalkylated
peptides (from the Lys(Me2) and Lys(Me/Et) peptides,
respectively; Figure S12). Therefore, it appears likely that
de-ethylation of Lys(Me/Et) by PHF8 is disfavored relative to
demethylation.
The results with PHF8 were largely mirrored in experi-
ments with the H3 Lysine-36(Me/Et) peptide and FBXL11
(Figure 2c, and Figures S5 and S13), indicating similar
reactivities for these two enzymes.
Overall, the results show that, when incorporated into
appropriate peptide sequences, Lys(Me/Et) is a substrate for
JMJD2E, PHF8, and FBXL11, producing monomethylated/
monoethylated and unalkylated products. The tri- (JMJD2E)
and di- (PHF8 and FBXL11) Ne-methyl demethylases display
With JMJD2E, product peaks were observed with masses
28 Da and 42 Da lower than the Lys(Me/Et) substrate
peptide, implying that both demethylation and de-ethylation
occur to form the monomethylated and unalkylated lysines
(Figure 2a). The assignments were supported by time course
studies using MS and NMR analysis (Figure 2j and Fig-
1
ure S9); H NMR analyses indicate the formation of formal-
dehyde (Figure S10) and acetaldehyde (Figure 3a). The latter
observation implies that de-ethylation occurs through hy-
droxylation adjacent to the Ne-ethyl lysyl amine, in an
analogous manner to demethylation (Scheme 1).
The major product of the reaction of H3 Lysine-9(Me/Et)
peptide with PHF8 was assigned as the unalkylated lysine
peptide, indicating that both demethylation and de-ethylation
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tion experiments with JMJD2E (Fig-
ure S18a), implying that both active-site
fit and/or bond strength may be factors
in determining the relative efficiency of
dealkylation. With PHF8, de-isopropy-
lation of the H3 Lysine-9(iPr) peptide
was also observed (Figure 2e); however,
no evidence for the formation of
a hydroxylated product was obtained.
Interestingly, in competition experi-
ments between the Lys(iPr) peptide
and the 14-mer Lys(Me1) peptide (see
above) with PHF8 the monomethylly-
sine substrate is preferred (Figure S18b),
which contrasts with the analogous
experiment with JMJD2E. No reaction
was observed between the H3 Lysine-
36(iPr) containing peptide and FBXL11,
revealing different selectivities across
the tested demethylases with respect to
the Lys(iPr) residue. We then analysed
the reactions of Ne-methyl-Ne-isopropyl-
lysine-containing peptides (Lys(Me/
iPr)) with the demethylases. Signifi-
cantly, the predominant product in sam-
ples of the H3 Lysine-9(Me/iPr) peptide
with JMJD2E possessed a mass 16 Da
higher than the starting peptide, imply-
ing hydroxylation (Figure 2 f). This
Figure 3. NMR analyses of reactions catalysed by JmjC demethylase. a) 1H NMR spectra of
JMJD2E-catalyzed formation of acetaldehyde by reaction with ART-Lys(Me3)-QTAR-Lys(Me/Et)-
STGGKA. b) 1H NMR spectra of JMJD2E-catalyzed formation of acetone by reaction with ART-
Lys(Me3)-QTAR-Lys(iPr)-STGGKA. c) 1H-1H COSY and TOCSY spectra of a sample containing
ART-Lys(Me3)-QTAR-Lys(Me/iPr)-STGGKA and JMJD2E after 1 hour of reaction. Correlations
corresponding to the hydroxylated lysine fragment are highlighted.
1
assignment was supported by H NMR
analyses (Figure 3c and Figure S19).
Upon incubation under 18O2, MALDI-
TOF analyses revealed formation of
a species with a + 18 Da mass shift
different preferences for the ethyl and methyl groups. We
then investigated reactions of the H3 sequences containing
Ne-isopropyllysine at Lysine-9 and Lysine-36 (Lys(iPr)). With
JMJD2E, the H3 Lysine-9(iPr) peptide was observed to
undergo loss of the isopropyl group to form the unalkylated
lysine species (Figure 2d), as supported by 1H NMR analyses
(Figure S14). MS experiments also revealed the formation of
a low-level species with a mass 16 Da greater than that of the
substrate peptide. Although its relatively low concentration
precluded detailed characterization, it is likely that this
product results from hydroxylation on an isopropyl CH3
(Figure S20), further supporting oxygenase-catalyzed hydrox-
ylation. 1H-1H COSY and TOCSY NMR analyses imply that
hydroxylation occurs on an isopropyl CH3 group (Figure 3c),
that is, two carbon atoms from the e-amine. However,
evidence for loss of the isopropyl group (and subsequent
loss of the methyl group) was apparent in the MALDI-TOF
spectra at low levels, indicating that oxidation of the isopropyl
group adjacent to the e-amine also occurs (trace levels of
acetone were also detected in the NMR experiments, Fig-
ure S19). No reaction was observed in samples containing the
H3 Lysine-9(Me/iPr) peptide and PHF8. However, the
samples with FBXL11 showed apparent demethylation of
the Ne-methyl group in MS and NMR analyses (Figure 2 g and
Figures S7 and S21). This observation suggests that the more
bulky isopropyl group is preferentially orientated away from
the catalytic iron in the FBXL11 active site; that is, the
selectivity differs from that of JMJD2E, where hydroxylation
is observed.
1
group. The H NMR spectrum of the reaction mixture after
60 min at 258C displayed a singlet resonance at dH =
2.17 ppm, which was assigned to the methyl protons of
1
acetone by both H and 13C chemical shift analysis and by
addition of a standard (Figure 3b and Figure S16). A small
singlet resonance was also observed at dH = 2.09 ppm, which
was tentatively assigned to the methyl protons of a-hydrox-
yacetone by 1H chemical shift analysis and by the addition of
a standard (Figure S15). Therefore, it is probable that
hydroxylation (and subsequent de-isoproylation) at the
isopropyl CH can occur after hydroxylation on the isopropyl
CH3. Notably, the Lys(iPr) substrate reacted more efficiently
than the analogous Lys(Me1)-containing peptide (14-mer,
ART-Lys(Me3)-QTAR-Lys(Me1)-STGGK) during competi-
Reactions with alkylated ornithine derivatives were then
investigated. The Nd-dimethylornithine peptide (H3 Orni-
thine-9(Me2)) did not react with JMJD2E; however, MS
activity assays with this peptide and PHF8 showed apparent
demethylation to form the corresponding Nd-monomethylor-
nithine peptide (H3 Ornithine-9(Me1), Figure 2h and Figur-
es S8a and S22). These findings demonstrate that PHF8 is
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identified on histones in cells.[18–20]
No reactions were observed for
any of the peptides with the deme-
thylases, suggesting that the tested
enzymes do not modify such spe-
cies, at least not with the sequen-
ces/conditions tested.
The relative reaction proficien-
cies of the analogues found to be
substrates were determined and
compared (Table S1). For all
three enzymes, Lys(Me/Et) was
the preferred substrate analogue;
however, all analogues were
weaker substrates than the analo-
gous Lys(Me2) peptides. Kinetic
parameters for the Lys(Me/Et),
Lys(iPr), and Lys(Me/iPr) peptides
with JMJD2E were also deter-
mined. KM, Vmax, and kcat values
for the Lys(Me/Et) peptide were
similar to those attained for the
corresponding Lys(Me2) peptide
(Table S2). The Lys(iPr) and Lys-
(Me/iPr) peptides showed higher
KM values (284.2 mm and 164.1 mm,
respectively, relative to 29.6 mm
and 42.7 mm for the Lys(Me2) and
Lys(Me/Et) peptides, respectively;
Table S2). Interestingly, the Vmax
and kcat values for the Lys(iPr)
peptide (Vmax = 0.152 mm sÀ1, kcat
=
Scheme 1. Summary of observed dealkylation/hydroxylation reactions catalyzed by JmjC demethylase.
0.303 sÀ1) were higher than values
for the other three peptides,
although the Lys(iPr) peptide is
able to accept shortened lysine analogues, whereas JMJD2E
does not; this observation correlates with X-ray analyses of
JMJD2A (a close homologue of JMJD2E) and PHF8 with
their respective H3K9 substrates (PDB codes 2OQ6 and
3KV4 respectively, Figure S25).[15,16] In these structures, the
methylated amine side chain at H3K9 apparently penetrates
deeper towards the catalytic iron in PHF8 than in JMJD2A,
implying that the shorter ornithine substrate may still bind
sufficiently near the iron for oxidation to occur. FBXL11 also
catalyzes demethylation of Nd-dimethylornithine when incor-
porated in an H3 Lysine-36 peptide (Figure 2i); this observa-
tion is consistent with the closer structural similarity of
FBXL11 to PHF8 than to JMJD2E. Finally, no reactions, at
least to give sufficient amounts of material for character-
ization, were observed for Nd-diethylornithine peptides (Orn-
(Et2)) with the three demethylases. This observation suggests
that the diethylamine moiety is too bulky to fit productively
into the active sites of the three enzymes; there was some MS-
based evidence that Orn(Et2) peptide is hydroxylated by
JMJD2E, although the low level of conversion precluded
characterization. The demethylases were also tested with
three N-acylated lysines; Ne-formyllysine (Lys(For)), Ne-
acetyllysine (Lys(Ac)), and Ne-crotonyllysine (Lys(Cr)),
respectively. All of these modifying groups have been
a weaker substrate than the Lys(Me/Et) and Lys(Me2)
peptides in competition experiments. This could reflect
faster product release for the reaction with the Lys(iPr)
À
peptide, or that oxidation of the weaker tertiary isopropyl C
H bond is faster than oxidation of the primary and secondary
À
C H bonds in the other substrates.
Overall, our results clearly indicate the potential of JmjC
enzymes to act on N-alkyl groups other than methyl. They
also imply substrate selectivity may be determined both by
À
active-site fit and C H bond strengths. The observation that
in addition to catalyzing N-dealkylation, JMJD2E can
catalyze formation of a stable alcohol strongly supports the
proposal of a hemiaminal intermediate during demethylation,
and a close evolutionary relationship between the JmjC
demethylases and related hydroxylases. Factor inhibiting
hypoxia inducible factor (FIH), a transcription factor hydrox-
ylase closely related to the JmjC demethylases, has been
found to catalyze hydroxylation not only of asparagine
residues (as in its action on hypoxia inducible factor), but
also on aspartate and histidine residues present in ankyrin
repeat proteins.[21,22] Thus, it is possible that the selectivity of
the JmjC enzymes (including those with as yet unassigned
functions) is even wider than suggested by our current work.
Although the physiological significance of our results is
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Keywords: demethylation · epigenetics · histone · hydroxylation ·
methyllysine
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