Angewandte
Chemie
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
Angew. Chem. Int. Ed. 2013, 52, 7709 –7713
ꢀ 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
7711