Journal of Medicinal Chemistry
BRIEF ARTICLE
of kcat but a much higher Km. The same cofactor change for the
wild-type vSET causes a 28-fold decrease of catalytic efficiency
but surprisingly a 1.5-fold decrease of Km. The decrease in Km
could be explained by the contribution of fortuitous interaction
between the benzyl group and some protein residues. The
extremely high Km for Y109G/16a might be associated with
the entropic penalty of fixing the benzyl group at a specific
orientation for its insertion into the artificial cavity. Thus, we
postulated that a second benzyl group at the 20-position might
restrict the conformation of 30-benzyl, securing its insertion into
the cavity. An additional 20-benzyl group in SAM (i.e., 20,30-
dibenzyl-SAM 16c) brings little change of the kcat/Km to the
wild-type vSET with an increase in kcat and Km. But a very
different scenario was observed for Y109G: a slight increase of
kcat but a much-improved Km (Table 2).
methyltransferase vSET and SAM. We selected the common N6-
position of the adenine ring and the novel 20- and 30-hydroxyl
groups of the ribose as the modification sites. We synthesized
seven SAM analogues and characterized their activity with the
wild-type and variant vSET proteins. We succeeded in engineer-
ing a paired vSET-L116A/16c, which is only slightly less active
than vSET-L116A/SAM but more active than the wild-type
vSET/16c. Given the presence of SAM in cells, further optimiza-
tion via chemical modifications of the enzyme and the cofactor is
needed to generate a paired vSET variant/modified SAM with
activity better than that of the variant with SAM. In summary, our
study demonstrates the feasibility of developing a methyl donor
cofactor that controls a histone lysine methyltransferase, which
could be a useful tool to study the effects of histone H3K27
methylation in vivo.
To mimic the hydrogen bond between Tyr109 and the 30-
hydroxyl of SAM, we synthesized another SAM analogue 19,23
which bears a carbamoylmethyl substituent at 20- and 30-oxygen
atoms, and tested its activity with Y109H, Y109D, or Y109N
(Supporting Information Scheme 1). We postulated that the
amide moiety of the carbamoylmethyl substituent of SAM could
form a hydrogen bond with the side chain on the residue
replacing Tyr109 in these variants. However, no activity was
detected for any of these vSET variants after an overnight
incubation and neither was any of these variants active with
SAM or the wild-type vSET with 19. These results emphasize the
importance of Tyr109 in substrate binding, and thus, change of
Tyr109 to His, Asp, or Asn is detrimental to vSET activity.
Inspection of residues around the ribose moiety of SAH in the
crystal structure suggests that changing Leu116 could create a
docking site for a benzyl group at the 20- or 30-position (Figure 1).
Indeed, most of the H3 peptide was converted to mono- or
dimethylated species after 19 h of reaction for L116A/16a
(Figure 2F). The variant was even more active with 16c, as
almost all the H3 peptide was converted to trimethylated species
(Figure 2G). Kinetic studies further revealed a kcat of 7.4 minꢀ1
and Km of 60 μM for L116A/SAM, which notably is even better
than the wild-type vSET/SAM pair (Table 2). When 16a was
used, the kcat of L116A was dropped to 0.15 minꢀ1, accompanied
by a slight increase of Km. Importantly, L116A/16c was more
active than WT/16c, with a 3.5-fold higher kcat and a 2.4-fold
lower Km. Comparison of the kcat/Km of L116A/16c with that of
L116A/16a argues that the presence of the benzyl groups at 20
and 30 positions has a synergistic effect on L116A catalytic
efficiency when compared to the single benzyl group analogues.
Since the presence of the 30-benzyl in SAM resulted in a
reduction in the enzymatic activity of the wild-type vSET and
L116A variant compared to SAM, we investigated whether 16b
would be more potent for L116A. The results show that for the
wild-type vSET, the removal of the 30-benzyl in 16c causes a
small decrease in kcat and Km with a net result of a 1.4-fold
decrease in the kcat/Km. However, for L116A, a 13-fold decrease
of kcat was seen with an overall 19-fold decrease in kcat/Km.
Interestingly, while adding a single benzyl to the 20- or 30-position
in SAM causes an ∼120-fold decrease in kcat/Km for L116A,
these two benzyl groups in 16c likely act synergistically, leading
to only a 7-fold decrease of kcat/Km.
’ EXPERIMENTAL SECTION
Chemical Synthesis. All compounds were synthesized using
commercially available starting materials without further purification
unless otherwise stated. 1H and 13C NMR spectra were recorded on a
Bruker 600 MHz NMR spectrometer using the residual signal of the
deuterated solvent as internal standard. Chemical shifts (δ) are reported
in ppm, coupling constants (J) in hertz. MS (ESI) analysis for new
compounds was performed on an Agilent G1969A high-resolution mass
spectrometer. All compounds that were tested in the biological assays
were analyzed by HPLC and LCMS to confirm the purity, which was
g95%. R and S indicate relative configurations.
(()-20,30-Dibenzyl-S-adenosylmethionine Iodide (16c). MeI
(0.03 mL, 0.5mmol) was added toa solution of15c (22.6mg, 0.04 mmol)
in HCOOH (1 mL). After the addition was completed, the mixture was
stirred at room temperature in the dark for 4 days. Isolation of 16c (6.0
mg, 21.3% yield) was achieved by HPLC on an Agilent Eclipse XDB-C18
9.4 mm ꢁ 250 mm column. 1H NMR (600 MHz, D2O) δ 2.22ꢀ2.26 (m,
1H), 2.31ꢀ2.35 (m, 1H), 2.83 (s, 3H), 2.95 (s, 2H), 3.45 (t, J = 9.0 Hz,
1H), 3.51ꢀ3.56 (m, 2H), 3.60ꢀ3.67 (m, 1H), 3.70ꢀ3.74 (m, 1H), 3.80
(t, J = 9.0 Hz, 1H), 3.82 (d, J = 12.0 Hz, 1H), 3.88 (d, J = 13.6 Hz, 1H),
4.14ꢀ4.23 (m, 2H), 4.34 (d, J = 13.6 Hz, 1H), 4.41 (s, 1H), 4.56ꢀ4.57
(m, 1H), 5.89 (d, J = 8.8 Hz, 1H), 6.72ꢀ6.78 (m, 1H), 6.86 (t, J = 7.9 Hz,
2H), 6.94 (d, J = 5.6 Hz, 2H), 7.42 (t, J = 5.3 Hz, 1H), 7.46 (t, J = 7.53 Hz,
2H), 7.50 (d, J = 3.9 Hz, 2H), 8.00 (s, 1H), 8.04 (s, 1H); HRMS (ESI)
calculated for C29H35N6O5S (M)+ 579.2384, found 579.2338.
Cloning, Expression, and Purification of vSET. vSET was
cloned into pET-22b and expressed as a nonfusion protein in BL21
(DE3) cells.8 The protein was purified with a heparin column and then a
Superdex 75 column.
Site-Directed Mutagenesis. Variants of vSET were generated
with the QuikChange mutagenesis kit (Stratagene). The presence of
appropriate amino acid change was confirmed by DNA sequencing. All
variants were purified as described for the wild type vSET.
Lysine Methyltransferase Reaction. The activity of a paired
vSET/cofactor was measured in a solution containing 1 μM enzyme,
100 μM H3 peptide (residues 13ꢀ33), and 1 mM methyl donor
cofactor. The reaction buffer consisted of 20 mM Tris at pH 8,
20 mM potassium chloride, and 10 mM magnesium chloride. The
reaction was performed at room temperature and stopped at a specified
time by the addition of trifluoroacetic acid (to 1%). The products were
analyzed by mass spectrometry.
Histone lysine methylation is a fundamental mechanism for
epigenetic control of gene expression in chromatin. Chemical
modulators capable of controlling the enzymatic activity of
specific lysine methyltransferases are extremely valuable tools.
In this study, we illustrated our approach with an H3K27-specific
’ ASSOCIATED CONTENT
S
Supporting Information. Synthesis procedures, spectro-
b
scopic details of the compounds, and enzyme kinetic analysis.
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dx.doi.org/10.1021/jm201000j |J. Med. Chem. 2011, 54, 7734–7738