Journal of the American Chemical Society
Article
MoaA-Catalyzed Reaction with 2′-ChloroGTP 20. 2′-ChloroGMP
(3 mM) was synthesized and phosphorylated using NDP kinase (100
units), guanylate kinase (50 μM), and ATP (10 mM). Guanylate
kinase was overexpressed and purified as described earlier.5 This crude
reaction mixture was used as a source of 2′-chloroGTP. For the MoaA-
catalyzed reaction, 250 μM MoaA, 2 mM 2′-chloroGTP, 3 mM
AdoMet, and 10 mM dithionite were mixed and incubated in the
anaerobic chamber for 5 h. Controls were set up for this reaction in
which either MoaA, AdoMet, dithionite, or NDP kinase/Guanylate
kinase were absent. The small molecule pool was analyzed by LC−MS.
For the trapping of the reactive 2′-Cl-ribose-derived product, all the
above reactions were performed in dithiothreitol (DTT) free buffer. A
50 μL portion of the small molecule pool was treated with 3 μL of
alkaline phosphatase followed by 3-mercaptobenzoic acid (50 μL of
100 mM solution) at 70 °C for 1 h.
MoaA-Catalyzed Reaction with 2′-DeoxyGTP (30b). MoaA (250
μM), 2 mM 2′-deoxyGTP, 3 mM AdoMet and 10 mM dithionite were
mixed and incubated in the anaerobic chamber for 5h at room
temperature. Controls were also set up in which MoaA, 2′-deoxyGTP,
AdoMet or dithionite were absent. The protein was removed by
ultrafiltration using a 10 kDa cutoff filter. The small molecule pool was
treated with 3 μL of alkaline phosphatases in the presence of 1 mM
MgCl2 and incubated in the anaerobic chamber for 3 h. The reaction
mixture was analyzed by LC−MS and the product was purified by
reverse phase HPLC. The isolated product was concentrated using a
vacuum centrifuge. For the coelution experiment 100 μM stock
solutions of compounds 25 and 26 were made. Standards (50 μL) and
50 μL of the concentrated product were mixed and analyzed by
HPLC.
Synthesis of Compounds 25, 26, and 2′-ChloroGTP 20. The
synthetic schemes and synthetic procedures for 2′-chloro GMP (the
2′R and the 2′S isomer), compound 25 and 26 are described in the
Supporting Information.
Figure 1. First steps in molybdopterin biosynthesis: (A) The carbon-
labeling pattern for the conversion of GTP 1 to cyclic pyranopterin
monophosphate 2. (B) Initial mechanistic proposal for the MoaA/
MoaC-catalyzed reaction.5,6
RESULTS
■
Characterization of the MoaA Reaction Product. A
time course for the product formation in the MoaA-catalyzed
reaction is shown in Supporting Information, Figure SI38. Most
of the product (approximately 70%) is formed in the first 90
min and there is no observed lag phase. The reaction was
allowed to proceed for 5 h to maximize product formation. The
protein was then removed by ultrafiltration, and the small
molecule pool was treated with alkaline phosphatase to
facilitate product purification by reverse phase HPLC. As the
reaction product is highly oxygen sensitive, undergoing
decomposition to a mixture of products, it was cleanly oxidized
using KI/I2 before exposure to air.5,8,9 The resulting product
was purified by HPLC as a fluorescent compound eluting at
17.4 min (Figure 2A). The UV−visible spectrum of the purified
product shows the characteristic features of a pterin (Figure
2B).2,10 Compound 25 was used as a standard to estimate the
yield of product 15 formation at 30−34% (75−85 μM of
product formed using 250 μM MoaA, assuming a single
turnover enzyme). Multiple small-scale MoaA reactions were
run to give sufficient product for characterization by LC−MS
and NMR spectroscopy.
Isolation of the Product of the MoaA-Catalyzed Reaction. The
reaction mixture consisted of 250 μM MoaA, 2 mM GTP, 3 mM
AdoMet, and 10 mM dithionite and was incubated in an anaerobic
chamber for 5 h at room temperature. The protein was then removed
by ultrafiltration using a 10 kDa cutoff filter. The resulting small
molecule pool was treated with 3 μL of alkaline phosphatases in the
presence of 1 mM MgCl2, incubated in the anaerobic chamber for an
additional 3 h and quenched with 100 μL of oxygen-free KI/I2 (5% I2
(w/v) and 10% KI (w/v) in water).5,8,9 The reaction mixture was then
purified by HPLC. The fluorescent product eluting at 17 min was
collected and dried using a vacuum centrifuge. Several such reaction
mixtures were purified to yield sufficient product for NMR
characterization. The dried samples were dissolved in 250 μL of a
90%:10% H2O/D2O mixture and analyzed by NMR (Bruker, 500
MHz).
MoaA reactions were also performed with MoaA overexpressed and
purified from the E. coli−MoaC deletion strain. An identical
fluorescent compound eluting at 17 min was observed (Supporting
Information, Figure SI 36).
Hydroxylamine Derivatization of the Reaction Product. The
reaction mixture consisted of 250 μM MoaA, 2 mM GTP, AdoMet,
and 10 mM dithionite and was incubated in an anaerobic chamber for
5 h. The protein was then removed by ultrafiltration using a 10 kDa
cutoff filter. The resulting small molecule pool was treated with 3 μL of
alkaline phosphatase in the presence of 1 mM MgCl2, incubated in the
anaerobic chamber for an additional 3 h and quenched with 100 μL of
oxygen free KI/I2 (5% I2 (w/v) and 10% KI (w/v) in water).5,8,9
PFBHA (100 μL of 40 mM) was then added, and the mixture was
heated at 65 °C for 1.5 h and analyzed by LC−MS. Control reactions,
lacking MoaA, GTP, SAM, or dithionite were also run and similarly
analyzed.
The LC−MS shows that the [M + H]+ (280.1 Da)
corresponds to the mass of compound 16 and 18 (Figure
1
2C). The H NMR and the dqfCOSY and HSQC for the
purified compound are shown in Figure 2D,E and Supporting
Information, Figure S35. All are consistent with a mixture of
compounds 16, 17, and 18. The spectra are not consistent with
an oxidation product derived from 8.7
Hydroxylamine Derivatization of the MoaA Reaction
Product. The oxidized reaction mixture prepared as described
above was treated with PFBHA and analyzed for oxime
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dx.doi.org/10.1021/ja502663k | J. Am. Chem. Soc. 2014, 136, 10609−10614