Journal of the American Chemical Society
Article
by sodium dithionite (10 mM, Kanto Chemical) for 1 h at room
temperature. Protein concentrations were determined with the
Bradford assay (Bio-Rad) with BSA as a standard or Nano Drop
Lite (Thermo Scientific) to estimate a concentration of NeoN (1 unit
at A280 as 1 mg/mL). UV/vis absorption spectra of NeoN were
determined by using a Shimadzu UV-2450 spectrophotometer.
Enzymes were freshly prepared for each set of experiments.
ESI-MS instrument [Shimadzu LCMS-2010EV mass spectrometer
equipped with LC-20AD pump, CTO-20A column oven, and SPD-
M20AUV detector, or LCQ (Finnigan) mass spectrometer equipped
with Shiseido semi-micro pump SI-1/2001, UV detector SI-1/2002,
and SSC-2300 column oven (Senshu)]. A TSKgel ODS-100Z column
(3 μm, 2.0 × 150 mm, TOSOH) was used for separation. Elution was
performed with 60% CH3CN for 10 min and then a linear gradient to
75% CH3CN for 20 min at a flow rate of 0.1 mL/min at 40 °C,
monitored at 350 nm. The negative mode was selected to detect DNP
derivatives of aminoglycosides.
Enzymatic Reaction of NeoN. The reduced NeoN (150 μM) was
incubated with 1 mM neomycin C (prepared enzymatically from
ribostamycin with NeoFDQB6) and 2 mM SAM (Sigma-Aldrich) in
buffer A in the presence of 10 mM sodium dithionite at room
temperature for 1 h. The reaction was quenched by addition of the
same volume of ethanol. The quenched reaction solution (20 μL) was
treated with 20 μL of 5% 1-fluoro-2,4-dinitrobenzene (Kanto
Chemical) in methanol, 10 μL of DMSO, and 2 μL of 2 M NaOH
at 60 °C for 1.5 h. The DNP derivatives of aminoglycosides were
extracted with 500 μL of ethyl acetate, and the organic layers were
evaporated. The residue was dissolved in 25 μL of CH3CN, and an
aliquot (5 μL) of the solution was injected into a HPLC (Hitachi
L7100 pump, L-7405-UV detector, L-7300 column oven or L-6250
pump, L-4000 UV detector, L-7300 column oven) equipped with a
TSKgel ODS-100Z column (5 μm, 4.6 × 250 mm, TOSOH). Elution
was performed with 60% CH3CN for 10 min and then a linear
gradient to 75% CH3CN for 20 min at a flow rate of 0.9 mL/min at 40
°C, monitored at 350 nm. The standard curves were generated with
the authentic neomycin B and C [a mixture of 85:15 (Nacalai
Tesque); 0.05, 0.1, 0.2, 0.5, 1.0, and 2.0 mM] after DNP derivatiztion
so as to correlate peak area for the amount of neomycin B and C.
The formed 5′-deoxyadenosine was also detected by HPLC. An
aliquot (5 μL) of the quenched enzymatic solution was injected into a
HPLC (same instrument mentioned above) equipped with a TSKgel
ODS-100Z column (5 μm, 4.6 × 250 mm, TOSOH). Elution was
performed with 10% CH3CN for 20 min at a flow rate 0.9 mL/min at
40 °C, monitored at 254 nm. The standard curves were generated with
the authentic 5′-deoxyadenosine (Sigma-Aldrich, 0.05, 0.1, 0.2, 0.5, 1.0,
and 2.0 mM) so as to correlate peak area with the amount of 5′-
deoxyadenosine.
To isolate deuterium-labeled neomycin B for NMR analysis, the
reduced NeoN (100 μM) was incubated with 1 mM neomycin C (4.9
mg) and 2 mM SAM (8.5 mg) in 10 mL of buffer B in the presence of
10 mM sodium dithionite at room temperature for 12 h in a glovebox.
The reaction was quenched by addition of the same volume of ethanol.
After centrifugation to remove the precipitate, the solution was loaded
on a DOWEX AG1-X8 column (OH− form, 2 × 6 cm) to obtain an
unbound fraction that contains cationic compounds. The solution was
then loaded on an Amberlite CG-50 column (NH4+ form, 2 × 6 cm),
washed with 0.1 M of aqueous ammonia, and then eluted with 1 M of
aqueous ammonia to obtain a free form of neomycin B. After removal
of the solvent, the residue was dissolved in water and loaded on a
2−
DOWEX AG1-X8 column (SO4 form, 1 × 6 cm) to obtain the
1
2
sulfate salt of neomycin B as a white solid (4.9 mg). H, H, and 13C
NMR spectra were recorded with a Bruker DRX-500 spectrometer.
Deuterium oxide (Acros Organics, 99.8 at.% enriched) was used as an
NMR solvent. Chemical shifts are reported in δ values based on the
1
solvent signal (δH 4.65) as a reference. H NMR (500 MHz, D2O): δ
1.96 (q, J = 12.6 Hz, 1H, H-2-ax), 2.42 (dt, J = 4.3, 12.7 Hz, 1H, H-2-
eq), 3.20 (dd, J = 5.9, 13.6 Hz, 1H, H-6′), 3.32 (d, J = 13.5 Hz, 1H, H-
6‴), 3.38 (d, J = 13.8 Hz, 1H, H-6‴), 3.40−3.50 (m, 4H, H-1, H-2′,
H-6′, H-3), 3.56 (t, J = 1.5 Hz 1H, H-2‴), 3.68−3.74(m, 2H, H-6, H-
5″), 3.79 (d, J = 2.2 Hz, 1H, H-4‴), 3.88 (dd, J = 2.8, 12.4 Hz, 1H, H-
5″), 3.91−3.96 (m, 2H, H-5, H-5′), 4.02 (t, J = 9.3 Hz, 1H, H-3′), 4.13
(t, J = 9.6 Hz, 1H, H-4), 4.18−4.22 (m, 2H, H-4″, H-3‴), 4.30 (m,
1H, H-5‴), 4.42 (dd, J = 1.6, 4.7 Hz, 1H, H-2″), 4.51 (dd, J = 4.9, 7.0
Hz, 1H, H-3″), 5.26 (d, J = 1.5 Hz, 1H, H-1‴), 5.40 (d, J = 1.8 Hz, 1H,
H-1″), 6.03 (d, J = 3.9 Hz, 1H, H-1′). 13C NMR (125 MHz, D2O): δ
28.4, 40.3, 40.4, 48.6, 50.0, 50.9, 53.7, 60.2, 67.1, 67.7, 68.1, 69.4, 71.0,
72.5, 73.3, 74.8, 75.4, 81.2, 84.9, 95.1, 95.1, 110.2.
Site-Directed Mutagenesis. The plasmid pET28-neoN was used
as a template DNA for PCR-based site-directed mutagenesis via the
QuikChange site-directed mutagenesis method. The oligonucleotides
listed in SI, Table S3 were used as primers. After confirmation of the
point mutation by DNA sequence analysis (Fasmac), the respective
resultant plasmids were introduced into E. coli C41(DE3) with
pRKSUF017. The recombinant mutant proteins of NeoN were
purified almost to homogeneity according to the above-mentioned
method for the wild type.
For the quantitative analysis of neomycin and 5′-deoxyadenosine
formations, NeoN (40 μM) was incubated with 0.5 mM neomycin C,
1.0 mM SAM in buffer A in the presence of 10 mM sodium dithionite
at room temperature for 10, 30, 60, 120, and 180 min and 15 h.
Enzymatic Reaction of NeoN with Neomycin Y2 (6‴-
Deamino-6‴-hydroxyneomycin C). The reduced NeoN (150
μM) was incubated with 1 mM neomycin Y2 (13 mg) and 2 mM
SAM (22.3 mg) in 10 mL of buffer A in the presence of 10 mM
sodium dithionite at room temperature for 12 h in a glovebox. The
formed neomycin Y1 (6‴-deamino-6‴-hydroxyneomycin C, 7.4 mg)
was purified according to the purification method for the deuterium-
1
labeled neomycin B mentioned below. H NMR (500 MHz, D2O): δ
1.65 (q, J = 12.5 Hz, 1H, H-2-ax), 2.24 (dt, J = 4.2, 12.7 Hz, 1H, H-2-
eq), 3.12−3.25 (m, 3H, H-4′, H-1, H-3), 3.29 (dd, J = 3.9, 10.8 Hz,
1H, H-6′), 3.34−3.41 (m, 2H, H-2′, H-6′), 3.46 (m, 1H, H-2‴), 3.58
(t, J = 8.9 Hz, 1H, H-6), 3.70 (m, 1H, H-4‴), 3.71−3.92 (m, 7H, H-
5″, H-6‴, H-6‴, H-4, H-5, H-5″, H-3′), 3.96 (ddd, J = 3.4, 8.5, 10.1
Hz, 1H, H-5′), 4.00 (m, 1H, H-5‴), 4.12−4.16 (m, 1H, H-3‴, H-4″),
4.32 (dd, J = 2.3, 4.9 Hz, 1H, H-2″), 4.41 (dd, J = 5.1 6.7 Hz, 1H, H-
3″), 5.14 (d, J = 1.6 Hz, 1H, H-1‴), 5.33 (d, J = 2.1 Hz, 1H, H-1″),
5.88 (d, J = 3.8 Hz, 1H, H-1′). 13C NMR (125 MHz, D2O): δ 30.4,
40.3, 48.8, 50.3, 51.3, 53.9, 60.4, 61.2, 66.7, 67.9, 68.7, 69.0, 71.0, 73.1,
73.7, 75.0, 75.6, 81.5, 81.5, 85.1, 95.4, 95.9, 109.9.
Enzymatic Reaction of NeoN in D2O-Containing Buffer. The
buffer of the reconstituted NeoN solution was exchanged with buffer B
[20 mM HEPES−NaOH, pD 7.8, 200 mM KCl, 10% glycerol
prepared with D2O (Acros Organics, 99.8 at.% enriched)] three times
during the ultracentrifugation with an Amicon Ultra-0.5 mL device.
The percentage of D2O in the buffer was estimated to be ca. 90%. The
obtained solution was reduced by sodium dithionite (10 mM) for 1 h
at room temperature and then used for the reaction. The other
reaction conditions were the same as mentioned above. An aliquot (2
μL) of DNP-derivativzed enzymatic solution was injected into an LC-
Enzymatic Reaction of C249A with Neomycin C. The reduced
C249A (150 μM) was incubated with 1 mM neomycin C and 2 mM
SAM in 10 mL of buffer A in the presence of 10 mM sodium dithionite
at room temperature for 30 h in a glovebox. The formed compound
(5.4 mg) was purified according to the purification method for the
deuterium-labeled neomycin B mentioned above. 1H NMR (500
MHz, D2O): δ 1.53 (q, J = 12.7 Hz, 1H, H-2-ax), 2.16 (dt, J = 4.2, 12.8
Hz, 1H, H-2-eq), 3.05 (m, 1H, H-3), 3.12 (d, J = 13.3 Hz, 1H, H-6‴),
3.12−3.18 (m, 2H, H-6′, H-1), 3.22 (dd, J = 3.7, 10.7 Hz, 1H, H-2′),
3.32 (d, J = 13.7 Hz, 1H, H-6‴), 3.33−3.40 (m, 2H, H-4′, H-6′), 3.51
(dd, J = 6.4, 9.0 Hz, 1H, H-2‴), 3.56 (t, J = 9.5 Hz, 1H, H-6), 3.60−
3.68 (m, 2H, H-4, H-5″), 3.70−3.85 (m, 4H, H-5, H-4‴, H-5″, H-3′),
3.93 (dd, J = 7.2, 9.2 Hz, 1H, H-3‴), 3.96 (m, 1H, H-5′), 4.04 (d, J =
6.1 Hz, 1H, H-1‴), 4.08−4.15 (m, 1H, H-4″, H-3″), 4.29 (dd, J = 2.5
4.1 Hz, 1H, H-2″), 5.33 (d, J = 2.3 Hz, 1H, H-1″), 5.79 (d, J = 4.3 Hz,
1H, H-1′). 13C NMR (125 MHz, D2O): δ 31.3, 40.3, 41.2, 48.9, 50.4,
54.0, 54.5, 60.9, 68.2, 69.0, 69.4, 71.0, 73.4, 73.4, 77.8, 78.9, 79.2, 79.4,
80.7, 81.1, 83.4, 85.1, 95.8, 109.8.
EPR Analysis. The reconstituted NeoN and C249A was reduced
with sodium dithionite (10 mM or 2 mM) at room temperature for 5−
30 min. Solutions of the reduced NeoN (300 μL, 300−600 μM) were
F
dx.doi.org/10.1021/ja507759f | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX