G. J. Poelarends et al.
1
1
a pre-packed PD-10 Sephadex G-25 gelfiltration column. The puri-
fied 4-OT proteins were assayed for residual tautomerase activity
using the spectrophotometric assay described above. To assess the
extent and specificity of the covalent labeling, the purified proteins
were also analyzed by ESI-MS.
The H NMR signals for trans-15 are as follows: H NMR (500 MHz,
20 mm phosphate/D O buffer, pH 7.3): d=6.70 (dd, J=8 Hz, 1H),
7.29 (m, 1H), 7.38 (m, 2H), 7.59 (d, J=6.5 Hz, 2H), 7.66 (d, J=
16 Hz, 1H), 9.44 ppm (d, J=8 Hz, 1H). Furthermore, in all samples
(with or without enzyme) the following extra signals were detected
after 1 week: H NMR (500 MHz, 20 mm phosphate/D O buffer,
2
1
2
Control reactions containing enzyme, buffer, and carbonyl com-
pH 7.3): d=1.91 (d, J=7.0 Hz, 3H), 6.07 (dd, J=8.0 Hz, 1H), 7.03
pound, or enzyme, buffer, and NaCNBH were carried out under
3
(
m, 1H), 9.22 ppm (d, J=8.5 Hz, 1H). These signals correspond to
the formation of 2-butenal (16). This self-condensation product of
is formed in slightly higher amounts in the incubations contain-
ing 4-OT as compared to the control reactions without enzyme.
Reference spectra of 3 (and its hydrate), 10, 15, and 16 are given
identical conditions. These mixtures did not lead to inactivation of
4
-OT.
3
Mass spectral analysis of modified 4-OT and peptide mapping:
[
4
2
1
-OT (0.5 mg) was incubated with 1 mm of acetaldehyde (3) in
in Figure S7].
0 mm NaH PO buffer (pH 7.3) for 1 h at 228C (total volume of
2
4
mL). A second 4-OT sample was not treated with 3 and was used
Detection of 15 by UV spectroscopy and GC/MS analysis: Al-
though 15 has characteristic H NMR spectroscopic signals, its
as the control sample. An aliquot from a 100 mm stock solution of
1
NaCNBH in H O was added to both samples to give a final con-
3
2
identity in the incubation mixture described above was further
confirmed by UV spectroscopy and GC/MS analysis. Accordingly,
after 14 d of incubation, a small aliquot was removed from the
mixture containing 3, 10, and 4-OT and diluted 200-fold in 20 mm
NaH PO buffer (pH 7.3). In addition, a small aliquot was removed
centration of 25 mm. After incubation for 1 h at 228C, the buffer
was exchanged against 10 mm NaH PO buffer (pH 8.0) by using a
2
4
pre-packed PD-10 sephadex G-25 gel-filtration column. The two
purified 4-OT proteins were assayed for tautomerase activity,
analyzed by ESI-MS, and used in the following peptide mapping
experiments.
2
4
from the control mixture (3 and 10 incubated without enzyme)
and also diluted 200-fold in the same buffer. Subsequently, UV/Vis
spectra were recorded from the diluted samples. Apart from the
characteristic absorbance peak of 10 (lmax =250 nm,
15 mm cm ), the sample containing 4-OT showed extra peaks at
lmax =290 nm (15, e290 =26.7 mm cm ) and around lmax =227 nm
(16, e227 =19 mm cm ). The absorbance peak corresponding to
15 was lacking in the control sample without enzyme.
For the peptide-mapping studies, unmodified 4-OT and 4-OT modi-
fied by 3 (50 mg) were vacuum-dried. The individual protein pellets
from the two samples were dissolved in 10m guanidine–HCl
e =
250
À1
À1
À1
À1
(
10 mL) and incubated for 2 h at 378C. Subsequently, the samples
À1
À1
were diluted with NH HCO3 buffer (90 mL, 100 mm; pH 8.0) and
4
incubated for 2 d at 378C with protease GluC (2.5 mL from a
À1
1
0 mgmL stock solution in H O). These two digested samples
2
For detection of 15 by GC/MS analysis, the remaining part (ca.
.6 mL) of the reaction mixture containing 3, 10, and 4-OT was re-
moved from the NMR tube and extracted with 1.8 mL of ethylace-
tate. The ethylacetate layer was dried over MgSO and subsequent-
ly analyzed by GC/MS. The control sample (3 and 10 incubated
without enzyme) was prepared and analyzed in the same way, but
did not show the presence of 15.
were analyzed by nano-LC–MS to identify the labeled peptide frag-
ment. Selected ions of both samples were subjected to MS/MS
analysis.
0
4
1
H NMR spectroscopic screening for carbonyl transformations by
4
-OT: A reaction mixture of 3 (50 mm) and either benzaldehyde
(10), acetophenone (11), cyclohexanecarboxyaldehyde (12) or
cyclopentanecarboxyaldehyde (13; each at ~50 mm) in NaH PO4
2
1
1
buffer (0.55 mL, 20 mm; pH 7.3) was placed in an NMR tube, along
H NMR spectroscopy assay for aldolase activity: H NMR spectra
À1
with D O (0.05 mL) and 4-OT (0.4 mg; 0.05 mL from a 8 mgmL
monitoring the aldol condensation of 3 with 10 catalyzed by either
wild-type 4-OT, L8R-4-OT, P1A-4-OT, R11A-4-OT, R39A-4-OT, 4-OT
inactivated by 3-BP, or synthetic 4-OT, were recorded as follows. In
an NMR tube, the enzyme (90 mm) was incubated with 3 and 10
2
solution). Similar mixtures without 4-OT (the control samples) were
also prepared to analyze the nonenzymatic (uncatalyzed) reaction.
1
H NMR spectra were recorded directly after mixing, and then after
1
, 7, and 14 d.
(50 mm each, unless stated otherwise) in 0.6 mL of 20 mm NaH PO
2 4
buffer (pH 7.3) at 228C. In addition, to each tube 0.05 mL of D O
was added. In two additional control experiments, 3 and 10 were
2
1
Compound 3 and its hydrate: H NMR (500 MHz, 20 mm phos-
phate/D O buffer, pH 7.3): d=1.19 (d, J=4.5 Hz, 3H), 2.09 (d, J=
.0 Hz, 3H), 5.11 (q, J=5.0 Hz, 1H), 9.52 ppm (q, J=3.0 Hz, 1H).
2
incubated without enzyme or with an aliquot from a mock purifi-
3
1
cation under otherwise identical conditions. The first H NMR spec-
1
trum was recorded immediately after mixing, and then after 7 and
Compound 10: H NMR (500 MHz, 20 mm phosphate/D O buffer,
2
1
4 days. The formation of 15 is indicative of the presence of aldo-
pH 7.3): d=7.46 (t, J=7.5 Hz, 2H), 7.60 (t, J=7.5 Hz, 1H), 7.79 (d,
J=8.0 Hz, 2H), 9.77 ppm (s, 1H).
1
lase activity. The H NMR signals for 3 (and its hydrate), 10 and 15
are described above.
1
Compound 11: H NMR (500 MHz, 20 mm phosphate/D O buffer,
2
Colorimetric assay for aldolase activity: Purified wild-type 4-OT,
pH 7.3): d=2.52 (s, 3H), 7.42 (t, J=7.5 Hz, 2H), 7.55 (t, J=7.5 Hz,
4
-OT mutants, and synthetic 4-OT were assayed for aldolase activity
1
H), 7.85 ppm (d, J=8.5 Hz, 2H).
by monitoring production of cinnamaldehyde (15) upon incuba-
tion with acetaldehyde (3) and benzaldehyde (10). Accordingly,
wild-type 4-OT, P1A-4-OT, R11A-4-OT, R39A-4-OT, L8R-4-OT, or syn-
thetic 4-OT (200 mg) were incubated (in separate vials) with 3 and
1
Compound 12: H NMR (500 MHz, 20 mm phosphate/D O buffer,
2
pH 7.3): d=1.06–1.40 (m, 6H), 1.50–1.70 (m, 4H), 1.77 (m, 1H),
.41 ppm (s, 1H).
9
1
10 (30 mm each) in NaH
PO buffer (1.2 mL, 20 mm; pH 7.3) at
2 4
Compound 13: H NMR (500 MHz, 20 mm phosphate/D O buffer,
2
2
28C. In two separate control experiments, 3 and 10 were incubat-
pH 7.3): d=1.20–1.54 (m, 6H), 1.58–1.76 (m, 2H), 2.27 (m, 1H),
.45 ppm (d, J=2.0 Hz, 1H).
ed without enzyme or with an aliquot of a mock purification. After
incubation of the reaction mixtures at 228C for 3 d, a sample
(50 mL) was removed and mixed with 0.2% (w/v) phloroglucinol
(150 mL) in HCl/EtOH (25:75%, v/v). Compound 15 forms a short-
9
In the incubation containing 3, 10, and 4-OT, extra signals were de-
tected after 1 d, indicating the formation of cinnamaldehyde (15).
6
08
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ChemBioChem 2011, 12, 602 – 609