Identification of a cyclic nucleotide as a cryptic intermediate in molybdenum cofactor biosynthesis

Article abstract of DOI:10.1021/ja401781t

The molybdenum cofactor (Moco) is a redox cofactor found in all kingdoms of life, and its biosynthesis is essential for survival of many organisms, including humans. The first step of Moco biosynthesis is a unique transformation of guanosine 5′-triphosphate (GTP) into cyclic pyranopterin monophosphate (cPMP). In bacteria, MoaA and MoaC catalyze this transformation, although the specific functions of these enzymes were not fully understood. Here, we report the first isolation and structural characterization of a product of MoaA. This molecule was isolated under anaerobic conditions from a solution of MoaA incubated with GTP, S-adenosyl-l-methionine, and sodium dithionite in the absence of MoaC. Structural characterization by chemical derivatization, MS, and NMR spectroscopy suggested the structure of this molecule to be (8S)-3′,8-cyclo-7,8-dihydroguanosine 5′-triphosphate (3′,8-cH₂GTP). The isolated 3′,8-cH₂GTP was converted to cPMP by MoaC or its human homologue, MOCS1B, with high specificities (K_m < 0.060 μM and 0.79 ± 0.24 μM for MoaC and MOCS1B, respectively), suggesting the physiological relevance of 3′,8-cH₂GTP. These observations, in combination with some mechanistic studies of MoaA, unambiguously demonstrate that MoaA catalyzes a unique radical C-C bond formation reaction and that, in contrast to previous proposals, MoaC plays a major role in the complex rearrangement to generate the pyranopterin ring.

Full text of DOI:10.1021/ja401781t

Article
pubs.acs.org/JACS
Identification of a Cyclic Nucleotide as a Cryptic Intermediate in
Molybdenum Cofactor Biosynthesis
Bradley M. Hover,† Anna Loksztejn,† Anthony A. Ribeiro,‡ and Kenichi Yokoyama*,†
‡
†Department of Biochemistry, Duke NMR Spectroscopy Center, and Department of Radiology, Duke University Medical Center,
Durham, North Carolina 27710, United States
S
* Supporting Information
ABSTRACT: The molybdenum cofactor (Moco) is a redox cofactor found in all kingdoms of life, and its biosynthesis is
essential for survival of many organisms, including humans. The first step of Moco biosynthesis is a unique transformation of
guanosine 5′-triphosphate (GTP) into cyclic pyranopterin monophosphate (cPMP). In bacteria, MoaA and MoaC catalyze this
transformation, although the specific functions of these enzymes were not fully understood. Here, we report the first isolation and
structural characterization of a product of MoaA. This molecule was isolated under anaerobic conditions from a solution of MoaA
incubated with GTP, S-adenosyl-L-methionine, and sodium dithionite in the absence of MoaC. Structural characterization by
chemical derivatization, MS, and NMR spectroscopy suggested the structure of this molecule to be (8S)-3′,8-cyclo-7,8-
dihydroguanosine 5′-triphosphate (3′,8-cH2GTP). The isolated 3′,8-cH2GTP was converted to cPMP by MoaC or its human
homologue, MOCS1B, with high specificities (Km < 0.060 μM and 0.79 0.24 μM for MoaC and MOCS1B, respectively),
suggesting the physiological relevance of 3′,8-cH2GTP. These observations, in combination with some mechanistic studies of
MoaA, unambiguously demonstrate that MoaA catalyzes a unique radical C−C bond formation reaction and that, in contrast to
previous proposals, MoaC plays a major role in the complex rearrangement to generate the pyranopterin ring.
C-8 of GTP is released as a formate during the reaction
catalyzed by GTP cyclohydrolases (Figure S1a).18,19 Thus, the
retention of GTP C-8 in Moco biosynthesis indicated a novel
mechanism of pterin ring formation.
INTRODUCTION
■
The molybdenum cofactor (Moco, 5, Figure 1a) is a redox
cofactor found in almost all organisms.1,2 Moco-dependent
enzymes play central roles in many biologically important
processes such as purine and sulfur catabolism in mammals,
anaerobic respiration in bacteria, and nitrate assimilation in
plants.2 In humans, Moco deficiency results in the pleiotropic
loss of all molybdenum enzyme activities, causing neurological
abnormalities and early childhood death.3,4 Unlike many other
cofactors, Moco cannot be taken up as a nutrient, and thus it
requires de novo biosynthesis. Moco biosynthesis (Figure 1a) is
thought to be conserved among all organisms5,6 and initiated by
the conversion of guanosine 5′-triphosphate (GTP, 1) into
cyclic pyranopterin monophosphate (cPMP, 3).7−9 cPMP is
then converted to Moco by the introduction of two sulfur
atoms10−12 and a molybdate.13−16
Two enzymes, MoaA and MoaC in bacteria, are responsible
for the conversion of GTP to cPMP.7,9,20,21 Bioinformatic
analysis suggested that MoaA belongs to the radical S-adenosyl-
L-methionine (SAM) superfamily.22 Enzymes in this super-
family catalyze the reductive cleavage of SAM using an oxygen-
labile [4Fe-4S] cluster as a reductant, and transiently generate a
5′-deoxyadenosyl radical (5′-dA•), which then abstracts a H-
atom from substrate to initiate radical reactions.23 The
classification of MoaA as a radical SAM enzyme was supported
by an in vitro activity assay of MoaA in the presence of MoaC,
in which the conversion of GTP into cPMP requires the
presence of SAM.9 The X-ray crystal structures of Staph-
ylococcus aureus MoaA9 revealed the binding of SAM to a [4Fe-
4S] cluster, in a fashion similar to other radical SAM
Previous isotope tracer experiments indicated that the
conversion of GTP to cPMP proceeds through the insertion
of C-8 of guanine between C-2′ and C-3′ of ribose (Figure
1a).7,17 This contrasts with the biosynthesis of pterin rings in
other GTP-derived cofactors such as folate and flavins, in which
Received: February 18, 2013
Published: April 29, 2013
© 2013 American Chemical Society
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Figure 1. (a) Moco biosynthetic pathway in bacteria and humans. The human enzymes are indicated in parentheses. The symbols on GTP and
cPMP indicate the source of the carbon and nitrogen atoms in cPMP as determined by isotope labeling studies.7,17 P designates a phosphate group.
3′,8-cH2GTP is shown in brackets, as it had not been identified prior to this report. (b) Previous proposal for the functions of MoaA and MoaC with
2-amino-5-formylamino-6-ribofuranosylamino-4-pyrimidinone triphosphate (6) as an intermediate.29,33 (c) Previous proposal for the functions of
MoaA and MoaC by Mehta et al.30 Pyranopterin triphosphate 7 was proposed as the product of MoaA.
enzymes.24,25 The structure also revealed the presence of an
additional C-terminal [4Fe-4S] cluster. This C-terminal [4Fe-
4S] cluster was shown to bind various purine nucleoside 5′-
triphosphates including GTP on the basis of equilibrium
dialysis and X-ray crystallography,26 as well as electron−nuclear
double-resonance (ENDOR) spectroscopy.27 Together with
the reported binding constant (0.29 μM),26 GTP was proposed
as a substrate of MoaA.
Many radical SAM enzymes are known to catalyze complex
rearrangement reactions.23,28 MoaA has also been considered to
catalyze the majority, if not all, of the complex rearrangement of
GTP to form the pyranopterin ring of cPMP.5,6,29,30 Schindelin
absence of MoaC, in which a molecule with light absorption at
320 nm and a mass signal at m/z = 524 [M+H]+ was observed.
Reactions using series of deuterated GTP suggested that a
deuterium at the 3′ position of GTP is transferred to 5′-
deoxyadenosine (5′-dA) produced in this assay. However, the
observed putative MoaA product was not isolated for further
structural characterization, and the relation of this observation
to the earlier one by Schindelin and Hanzelmann26 was not
̈
discussed. It is currently unknown whether the putative MoaA
product could serve as a substrate of MoaC and be converted to
cPMP. Nevertheless, on the basis of these observations, the
authors proposed that MoaA catalyzes conversion of GTP into
pyranopterin triphosphate (7, Figure 1c).30
and Hanzelmann first proposed that, in the absence of MoaC,
̈
purified MoaA catalyzes conversion of GTP into a molecule
with a 6-hydroxy-2,4,5-triaminopyrimidine partial structure, on
the basis of the chemical derivatization to dimethylpterin
(DMPT).26 Unfortunately, no data were presented. This
observation was analogous to those for folate biosynthesis,
where the imidazole moiety of guanine base in GTP is first
hydrolyzed to a 2-amino-5-formylamino-6-ribofuranosylamino-
4-pyrimidinone triphosphate (Figure S1a),31 which may be
chemically derivatized to DMPT (Figure S1b). This analogy
has prompted speculations about the reaction catalyzed by
MoaA,29,32 which consider 2-amino-5-formylamino-6-ribofur-
anosylamino-4-pyrimidinone triphosphate (6) as an intermedi-
ate or a product of the MoaA-catalyzed reaction (Figure 1b).
Recently, a more definitive proposal for the product of MoaA
was made by Mehta et al.30 In their report, the authors
performed LC-MS analysis of small molecules produced after
incubation of MoaA with GTP, SAM, and dithionite in the
The role of MoaC, on the other hand, has been significantly
under-appreciated. MoaC does not show significant amino acid
sequence similarities to functionally characterized proteins.
Currently, structures of MoaC from four different organisms
have been reported.32−35 A putative ligand binding site was
proposed on the basis of the conservation of amino acid
residues and their relative positions in a crystal structure.34
A
structure of MoaC from Thermus thermophilus in combination
with isothermal titration calorimetry experiments suggested
that nucleotide triphosphate may bind to the putative ligand
binding site.33 In the recent publication by Mehta et al.,30
MoaC was proposed to be responsible only for the formation of
the cyclic phosphate (Figure 1c) on the basis of their premise
that the MoaA product is pyranopterin triphosphate. However,
uncertainty remains about the relevance of this proposal due to
the limited characterization of the MoaA reaction product.
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initiated by the addition of MoaA and incubated for 60 min at 25 °C,
at which point MoaA was removed by ultrafiltration (Amicon Ultra 30
K, Millipore). To an aliquot (80 μL) of the resulting small-molecule
fraction, 10 μL of 50 μM MoaC was added and incubated for 60 min
at 25 °C. The reaction was stopped by addition of 10 μL of 25% (w/v)
trichloroacetic acid (TCA). cPMP formed during the incubation with
MoaC was oxidized to compound Z and quantified by HPLC
following the published protocol.7 Briefly, a solution (10 μL)
containing 1% (w/v) I2 and 2% (w/v) KI was added to the quenched
reaction mixture and incubated for 20 min at room temperature. After
removal of precipitate by centrifugation, an aliquot (10 μL) of the
supernatant was injected to the HPLC. Chromatography was
performed by an isocratic elution with 0.1% TFA in H2O with a
flow rate of 1 mL/min and monitored by fluorescence (excitation 367
nm, emission 450 nm). Under these conditions, the void volume was
1.5 min, and compound Z eluted at 2.7 min. The authentic compound
Z standard was isolated from ΔMoeB E. coli strain by following the
reported protocol.8
Detection of the MoaA Product as DMPT. MoaA (5 μM) was
incubated with GTP (1 mM), SAM (1 mM), and sodium dithionite (1
mM) in assay buffer (0.5 mL). The reaction was initiated by the
addition of MoaA and incubated at 25 °C for a specified time. An
aliquot (80 μL) was removed at each time point and mixed with 10 μL
of 0.5 M HCl to quench the reaction. The resulting mixture was
incubated at 95 °C for 5 min to facilitate the hydrolysis, followed by
addition of 6 μL of 1 M NaOH to adjust the pH to 8.5. The resulting
solution was combined with 25 μL of 0.66% (v/v) 2,3-butanedione
solution in 0.9 M Tris-HCl, pH 8.5, and incubated at 95 °C for 45
min. After removal of precipitate by centrifugation, an aliquot (10 μL)
of the supernatant was injected to the HPLC. Chromatography was
performed by an isocratic elution with 92.5% 20 mM sodium acetate,
pH 6.0, and 7.5% MeOH with a flow rate of 1 mL/min and monitored
by fluorescence (excitation 365 nm, emission 445 nm). Under these
conditions, the void volume was 1.5 min, and DMPT eluted at 5.6
min.
3′,8-cH2GTP Stability Test. The temperature and pH stabilities
were assessed by incubating 3′,8-cH2GTP (50 μM) solution in
anaerobic buffer (15 mM ammonium formate, pH 3.5, 50 mM Tris-
HCl, pH 7.6, or 15 mM ammonium bicarbonate, pH 9.0) in the
glovebox at 10 or 22 °C. To test for oxygen sensitivity, 3′,8-cH2GTP
(50 μM) solution was taken out of the glovebox and exposed to air by
rigorous pipetting. For each reaction, an aliquot (10 μL) was removed
at specified time points and incubated with MoaC in 80 μL of assay
buffer (50 mM Tris-HCl, pH 7.6, 0.3 M NaCl, 1 mM MgCl2, and 2
mM DTT). The reaction was quenched with 10 μL of 25% TCA, and
cPMP was quantified as described above for the stepwise assay.
Isolation of 3′,8-cH2GTP. The MoaA reaction and subsequent
purification of 3′,8-cH2GTP were carried out under strict anaerobic
conditions (<0.1 ppm O2). The large-scale MoaA reaction for the
isolation of 3′,8-cH2GTP was carried out with MoaA (200 μM), SAM
(1 mM), sodium dithionite (1 mM), and GTP or [U-13C5,15N10]GTP
(200 μM) in 50 mM Tris-HCl, pH 7.6 (200 mL), at 22 °C for 60 min.
These reactions typically produced 12−20 μmol of 3′,8-cH2GTP.
Subsequent purification was carried out at 10 °C. The MoaA reaction
mixture was first passed through an ultrafiltration membrane (Amicon
YM-30, Millipore) to remove the protein, and the resulting filtrate was
applied to a QAE A25 Sephadex (250 mL, bicarbonate form) column.
The column was washed with 750 mL of H2O, and elution was
performed by a linear gradient (600 × 600 mL) of 200−800 mM
ammonium bicarbonate, pH 9.1. To identify the fractions containing
3′,8-cH2GTP, an aliquot (10 μL) of each fraction was mixed with 80
μL of MoaC (20 μM) in assay buffer and incubated for 30 min at 25
°C. The resulting solutions were analyzed for cPMP as described
above for the stepwise assay. The fractions containing 3′,8-cH2GTP
were combined, lyophilized, re-dissolved in water, and applied to a
DEAE sepharose FF (15 mL, bicarbonate form) column. The column
was washed with 50 mL of 100 mM ammonium bicarbonate, pH 9.1,
and eluted by a linear gradient (300 × 300 mL) of 100−200 mM
ammonium bicarbonate, pH 9.1. The fractions containing 3′,8-
cH2GTP were identified by UV−vis absorption spectroscopy and
In the current study, we report the isolation and detailed
characterization of the MoaA reaction product, which
unambiguously delineated the individual reactions catalyzed
by MoaA and MoaC. Isolation of the MoaA reaction product
was achieved under anaerobic conditions with careful control of
pH. Structural characterization by chemical derivatization, MS,
and NMR spectroscopy established the structure of this
molecule as (8S)-3′,8-cyclo-7,8-dihydroguanosine 5′-triphos-
phate (3′,8-cH2GTP, 2). The relevance of the isolated 3′,8-
cH2GTP as a physiological Moco biosynthetic intermediate was
demonstrated by steady-state kinetic analysis of MoaC and its
human homologue, MOCS1B. MoaC and MOCS1B converted
3′,8-cH2GTP to cPMP with Km values of <0.06 and 0.79 μM,
respectively. Additional studies on the stoichiometry of the
MoaA reaction in combination with an isotope tracer
experiment suggest that the conversion of GTP to 3′,8-
cH2GTP proceeds through H-abstraction from the 3′ position
by consuming a stoichiometric amount of SAM. The
observations presented here in sum unequivocally delineate
the individual functions of MoaA and MoaC and provide
insights into the biosynthesis of cPMP. The current
identification of 3′,8-cH2GTP as a substrate of MoaC is a
sharp contrast to previous proposals,29,30 in which MoaC was
thought to have minimal or no function in the formation of the
pyranopterin structure. These results provide a basis for future
mechanistic studies of reactions catalyzed by these two
enzymes.
MATERIALS AND METHODS
■
QAE A25 Sephadex resin, guanosine 5′-triphosphate (GTP),
[U-13C5,15N10]GTP, S-adenosyl-L-methionine (SAM), 5′-deoxyades-
nosine (5′-dA), dithiothreitol (DTT), sodium dithionite, 2,3-
butanedione, and dimethylpterin (DMPT) were purchased from
Sigma-Aldrich. DEAE sepharose FF resin was from GE Healthcare.
[3-2H]Ribose was from Omicron Biochemicals Inc. Chemically
competent Escherichia coli DH5α and BL21(DE3) cells and all PCR
primers were from Invitrogen. pET expression plasmids were from
Novagen. Calf-intestine alkaline phosphatase (CIAP, 20 U/μL) was
from NEB. UV−vis absorption spectra were determined using a U-
3900 UV−vis ratio-recording double-beam spectrometer (Hitachi) or
a Nanodrop 1000 instrument (Thermo Scientific). Nonlinear least-
squares fitting of kinetic data was carried out using KaleidaGraph
software (Synergy Software, Reading, PA). Anaerobic experiments
were carried out in a UNIlab workstation glovebox (MBaun, Stratham,
NH) maintained at 10 2 °C with O2 concentration <0.1 ppm. All
anaerobic solutions were degassed on a Schlenk line and equilibrated
in the glovebox atmosphere for >12 h. All plastic devices for anaerobic
use were evacuated for >12 h in the glovebox antechamber before
being brought into the glovebox. All DNA sequences were confirmed
by Eton Bioscience Inc. PCR was carried out using PfuUltraII
polymerase (Stratagene) according to the manufacturer’s protocol. All
HPLC experiments were performed on a Hitachi L-2130 pump
equipped with an L-2455 diode array detector, an L-2485 fluorescence
detector, an L-2200 autosampler, and an ODS Hypersil C18 column
(Thermo Scientific) housed in an L-2300 column oven maintained at
40 °C. S. aureus MoaA and MoaC were expressed and purified by
published protocols9 with minor modification as described in the
Supporting Information. The expression plasmids for SUF proteins36
and MOCS1B37 were kindly provided by Dr. Herman Schindelin. The
5-methylthioribose kinase expression plasmid38 was a generous gift
from Dr. John Gerlt.
Stepwise Assay of MoaA and MoaC. The stepwise MoaA/
MoaC assays were carried out under anaerobic conditions. First, MoaA
(5 μM) was incubated with GTP (1 mM), SAM (1 mM), and sodium
dithionite (1 mM) in 200 μL of assay buffer (50 mM Tris-HCl, pH
7.6, 1 mM MgCl2, 2 mM DTT, and 0.3 M NaCl). The reaction was
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the MoaC activity assay described above. After removal of the solvent
by lyophilization, the purified 3′,8-cH2GTP was characterized by NMR
and ESI-TOF-MS.
induced with 0.3 mM IPTG. The culture was continued for an
additional 20 h until saturation, and cells were harvested by
centrifugation, frozen in liquid N2, and stored at −80 °C. Typically,
10 g of wet cell paste per liter was obtained.
Phosphate Quantitation. Phosphate quantitation was carried out
on the basis of the published protocol.8,39 Purified 3′,8-cH2GTP (4
pmol) was incubated with CIP (5 units; each unit is defined as the
amount of enzyme that hydrolyzes 1 nmol of p-nitrophenylphosphate
per minute at 37 °C) for 1 h at 37 °C in 100 μL of 50 mM Tris-HCl,
pH 7.9, containing 100 mM NaCl, 10 mM MgCl2, and 1 mM DTT.
The resulting solution was then mixed with 0.233 mL of 1.4% (w/v)
ascorbic acid and 0.36% (w/v) ammonium molybdate tetrahydrate in
0.86 M H2SO4. After an hour of incubation at 37 °C, the phosphate
concentration was determined on the basis of the absorption of the
phosphomolybdate complex at 820 nm (ε820nm = 26.0 mM−1 cm−1).39
Determination of the Molecular Weight of 3′,8-cH2GTP.
Purified 3′,8-cH2GTP (100 μM) in anaerobic water was prepared in
the glovebox. An aliquot (10 μL) of the sample was injected into the
ESI-TOF MS instrument (Agilent 6224) operated in the negative ion
mode. The typical mass accuracy of the instrument was 5 ppm. For the
characterization of the observed MS signal, 3′,8-cH2GTP (100 μM)
was anaerobically incubated with MoaC (50 μM) in 400 μL of 300
mM ammonium bicarbonate buffer, pH 9.1, for 20 min at 25 °C. After
removal of MoaC by ultrafiltration (Amicon YM-10, Millipore), the
solution was lyophilized, and the residue was dissolved in 400 μL of
anaerobic water. The resulting solution was analyzed by ESI-TOF MS
as described for 3′,8-cH2GTP.
NMR Measurements of Purified 3′,8-cH2GTP. All NMR spectra
were recorded on 500, 600, or 800 MHz Varian Inova NMR
spectrometers operated with VNMRJ 3.1 software and analyzed by the
ACD/NMR processor (ACD/Laboratories). The samples were
dissolved in anaerobic deuterium oxide (Sigma-Aldrich, 99.9 atom%
enriched), loaded in 3 mm NMR tubes (Wilmad LabGlass) in the
glovebox, and sealed with butyl rubber septa (Sigma-Aldrich). NMR
measurements were carried out for 24−48 h at a sample temperature
of 6 °C. No decomposition of 3′,8-cH2GTP was observed during the
NMR measurements based on a comparison of 1H NMR spectra at the
beginning and the end of the measurements. Chemical shifts are
reported in δ based on an internal standard, 4,4-dimethyl-4-
Purification of MOCS1B. The cell paste (10 g) of E. coli
BL21(DE3) expressing MOCS1B was suspended in 50 mL of buffer A
(50 mM Tris-HCl, pH 9.0, 0.3 M NaCl, 10% glycerol) supplemented
with 1% Triton-X and a protease inhibitor cocktail (Calbiochem no.
539132). The cell suspension was homogenized and lysed by two
passages through a French pressure cell operating at 14 000 psi. After
removal of cell debris by centrifugation (20000g, 20 min, 4 °C), DNA
was precipitated by dropwise addition of 0.2 vol of buffer A containing
8% (w/v) streptomycin sulfate. The mixture was stirred for an
additional 15 min, and the precipitated DNA was removed by
centrifugation (20000g, 20 min, 4 °C). Solid (NH4)2SO4 (0.23 g per
mL) was then added over 15 min to 40% saturation. The solution was
stirred for an additional 20 min, and the precipitated protein was
isolated by centrifugation (20 000g, 20 min, 4 °C). The protein pellet
was dissolved in a minimal volume of buffer A supplemented with 1%
Triton-X, 0.5 mM phenylmethanesulfonyl fluoride (PMSF), and 20
mM imidazole and applied to a Ni-NTA column (10 mL). The
column was then washed with 10 vol of the same buffer, followed by
another 10 vol of buffer A containing 20 mM imidazole. MOCS1B was
subsequently eluted with 500 mM imidazole in buffer A. Fractions
containing MOCS1B were identified by SDS-PAGE, combined, and
exchanged into buffer A on a Sephadex G-25 column. The resulting
MOCS1B was concentrated by ultrafiltration (Amicon Ultra 3 K,
Millipore) to 0.2 mM, flash frozen in liquid nitrogen, and stored at
−80 °C. The concentration of MOCS1B was determined on the basis
of UV absorption at 280 nm using an extinction coefficient (ε280nm
=
15.9 mM−1 cm−1) determined by Edelhoch’s method.40 To character-
ize the three MOCS1B polypeptides, each polypeptide was purified on
SDS-PAGE and digested with L-1-tosylamido-2-phenylethyl chlor-
omethyl ketone-treated Porcin trypsin (Promega). The resulting
tryptic peptides were analyzed by LC-MS at the Duke proteomics core
facility.
Activity Assays of MoaC and MOCS1B. MoaC (0.1 μM) or
MOCS1B (0.5 μM) was anaerobically incubated with 3′,8-cH2GTP at
specified concentrations in assay buffer at 25 °C. The reaction was
initiated by the addition of MoaC or MOCS1B, and an aliquot (90
μL) was removed at each time point and mixed with 10 μL of 25% (w/
v) TCA to quench the reaction. cPMP was quantified as described
above for the stepwise assay.
silapentane-1-sulfonic acid (DSS, δCH = 0.00) as reference. Maleic
3
acid (δCH = 6.0, Sigma-Aldrich) was used as an internal standard for
signal quantitation.
MoaA/C Coupled Assay. Enzyme activity assays were performed
at 25 °C under anaerobic conditions by incubating MoaA (0.5 μM)
and MoaC (5 μM) with GTP (1 mM), SAM (1 mM), and sodium
dithionite (1 mM) in assay buffer. The reaction was initiated by the
addition of MoaA. At each time point, an aliquot (90 μL) was removed
and mixed with 10 μL of 25% (w/v) TCA to quench the reaction.
cPMP was quantified as described above for the stepwise assay.
Preparation of MOCS1B Expression Plasmid. The MOCS1B
gene in the pQE-MOCS1B plasmid originally prepared by
Quantitation of 5′-dA. Enzyme reactions were performed and
quenched as described for the MoaA/C coupled assay except that 20
μM MoaA and 40 μM MoaC were used. After the TCA quenching, the
solution was clarified by centrifugation, and an aliquot (70 μL) of the
supernatant was injected to HPLC. Chromatography was performed
by a linear gradient of 0−15% MeOH in 27 mM KH2PO4, pH 4.5 (25
min, 2 mL/min), and monitored by UV absorption at 256 nm.
Preparation of [3′-2H]GTP. Enzymatic Synthesis of [3′-2H]-
Guanosine. [3-2H]Ribose (1 mM, > 95% enriched) was incubated at
27 °C with ATP (2 mM), guanine (2 mM), 5-methylthioribose kinase
(3.5 μM),38 and purine nucleoside phosphorylase (Sigma-Aldrich, 3.5
μM) in 200 mL of 10 mM Tris-HCl, pH 7.6, containing 2.5 mM
MgCl2 and 2.5 mM DTT. After 2 h of incubation at 27 °C, the
solution was adjusted to pH 3 with 1 M HCl and loaded on to a
Dowex 50W-X8 column (2 × 10 cm, ammonium form). The column
was washed with 15 mM ammonium formate, pH 3.0, and eluted with
300 mM ammonium formate, pH 9.0. Fractions containing guanosine
were identified by UV−vis absorption and thin-layer chromatography.
Guanosine was typically eluted after 7−10 column volumes of elution.
The fractions containing guanosine were combined, and the solvent
was removed by lyophilization to yield 100 μmol of [3′-2H]guanosine
(94 3 atom% 2H): 1H NMR (500 MHz, D2O) δ 7.95 (s, 1 H), 5.86
(d, J = 7.3 Hz, 1 H), 4.62 (m, 1H), 4.18 (br, 1 H), 3.84 (dd, J = 2.9,
12.7 Hz, 1H), 3.77 (dd, J = 3.9, 12.7 Hz, 1H); HRMS (ESI-TOF) m/z
[M+H]+ calcd for C10H13DN5O5 285.106, found 285.107.
Hanzelmann et al.37 was subcloned into pET-28b to improve protein
̈
expression. NdeI restriction site was introduced by PCR at the 5′ end
of the MOCS1B gene in the pQE-MOCS1B plasmid using primers
MOCS1B-f (CAT CAC CAT CAC CAT ATGATG AGT TTC TC)
and MOCS1B-r (GAG AAA CTC ATC ATA TGG TGA TGG TGA
TG). After confirmation of the DNA sequence, the resulting plasmid
was digested with NdeI and SalI, and the MOCS1B gene fragment was
subcloned into the corresponding site of pET28b to obtain pET-
MOCS1B.
Expression of MOCS1B. E. coli BL21(DE3) cells were co-
transformed with pET-MOCS1B and grown overnight on LB agar
plates. All growths were carried out in the presence of kanamycin (50
mg/L). A single colony was picked and grown in LB medium (5 mL)
to saturation (∼16 h). Two milliliters of this solution was diluted into
200 mL of LB medium in a 500 mL baffled flask and incubated at 37
°C until growth reached saturation. A portion of this culture (10 mL)
was then used to inoculate 1.5 L of 2YT medium in a 2.8 L baffled
flask and grown at 37 °C until OD600 reached 0.6−0.8, at which time
the temperature was lowered to 15 °C and MOCS1B expression was
Phosphorylation of [3′-2H]Guanosine. Phosphorylation of guano-
sine was performed by the Ludwig’s method.41 Briefly, [3′-2H]-
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guanosine (100 μmol) was dried by co-evaporation with dry pyridine
and dissolved in dry (MeO)3PO (1.5 mL). To this solution was added
POCl3 (130 μmol), and the mixture was stirred on ice for 2 h. Bis-tri-
n-butylammonium pyrophosphate (500 μmol) in anhydrous DMF (2
mL) and Bu3N (100 μL) was then added under rigorous stirring. After
10 min, 7 mL of 1 M triethylammonium bicarbonate, pH 7.5, was
poured into the solution and stirred for 15 min. The resulting mixture
was diluted by 5-fold with water and applied to QAE A25 Sephadex
(Sigma, 4 × 20 cm, bicarbonate form). The column was washed with
0.2 M ammonium bicarbonate pH 8.0 (100 mL), and the elution was
performed with a linear gradient (450 × 450 mL) of 0.2−1 M
ammonium bicarbonate, pH 8.0. GTP was eluted between 0.55 and
0.7 M ammonium bicarbonate, judged by UV−vis absorption.
Lyophilization of these fractions yielded 24 μmol of [3′-2H]GTP
(94 3 atom% 2H): 1H NMR (500 MHz, D2O) δ 7.92 (s, 1 H), 5.71
(d, J = 5.9 Hz, 1 H), 4.56 (d, J = 5.9 Hz, 1 H), 4.15 (br, 1 H), 4.04 (m,
2 H); 31P NMR (160 MHz, D2O) δ −8.04 (br), −11.26 (d, J = 19
Hz), −22.45 (br); HRMS (ESI-TOF) m/z [M−H]− calcd for
C10H14DN5O14P3 522.990, found 522.991.
MoaA Reaction with [3′-2H]GTP and Isolation of [5′-2H]5′-
dA. [3′-2H]GTP (98 μM) was anaerobically incubated with MoaA (50
μM), MoaC (100 μM), SAM (1 mM), and sodium dithionite (1 mM)
in assay buffer for 1 h at 25 °C. Proteins were removed by
ultrafiltration (Amicon Ultra 30 K, Millipore), and the resulting filtrate
was applied to a C18 RP Silica 90 column (Sigma, 2 × 8 cm)
equilibrated in water. Elution was performed using a linear gradient
(300 × 300 mL) with 0−30% MeOH in water. 5′-dA was eluted at
15% MeOH judged by UV−vis absorption at 256 nm. After removal of
the solvent by rotary evaporation, the sample was further purified by
HPLC using conditions identical to those described above for the
quantitation of 5′-dA, except that 30 mM ammonium formate, pH 4.5,
was used as the aqueous solvent. The resulting sample was lyophilized
1
2
and characterized by H NMR, H NMR, and ESI-TOF-MS.
RESULTS
■
Investigation for an Intermediate between GTP and
cPMP. While small molecules produced by MoaA were
previously proposed to serve as a substrate of MoaC,30 the
conversion of this molecule to cPMP by MoaC in the absence
of MoaA has never been demonstrated. Considering the
complexity of the conversion of GTP into cPMP, and enzyme-
catalyzed radical reactions in general,28,42 the absence of such
demonstration leaves significant ambiguity about the relevancy
of the observations to Moco biosynthesis. Thus, we carried out
stepwise activity assays using S. aureus MoaA and MoaC. In
these assays, GTP (1 mM) was first incubated with MoaA (5
μM), SAM (1 mM), and sodium dithionite (1 mM) in the
absence of MoaC. The resulting small molecules were
separated from MoaA by ultrafiltration, and subsequently
incubated with MoaC (5.6 μM). Any cPMP present in the
resulting reaction solution was quantified by HPLC analysis
after a conversion to its fluorescent derivative, compound Z (8,
Figure 2a), following the previously established protocol.8
Figure 2a shows the results of HPLC analysis that shows a peak
co-migrating with compound Z. Quantitation of the observed
HPLC peak using an authentic standard suggested formation of
Figure 2. Activity assays of MoaA. (a) HPLC analysis of stepwise
MoaA/MoaC activity assay (excitation 367 nm, emission 450 nm). In
the complete reaction, GTP (1 mM), SAM (1 mM) and sodium
dithionite (1 mM) were incubated with MoaA (5 μM) for 60 min at
25 °C, followed by removal of MoaA by ultrafiltration and incubation
with MoaC (15 μM) for 60 min at 25 °C. cPMP was converted to
compound Z (8), and detected by HPLC. Also shown are the
chromatograms for the compound Z standard, and control reactions
without GTP or SAM. (b) HPLC analysis of MoaA activity assay
(excitation 365 nm, emission 445 nm). In the complete condition,
MoaA (5 μM), was incubated with GTP (1 mM), SAM (1 mM), and
sodium dithionite (1 mM) at 25 °C for 60 min. The reaction product
was subjected to acid hydrolysis, followed by incubation with 2,3-
3.6
0.3 μM compound Z, substoichiometric to MoaA.
Control reactions lacking GTP or SAM did not yield
compound Z. These observations are consistent with the
presence of a small molecule intermediate that is produced by
MoaA, and converted to cPMP by MoaC.
Chemical Derivatization of the MoaA Reaction
Product. For the biosynthesis of other pterins and flavins,
nucleotide biosynthetic intermediates with a 6-hydroxy-2,4,5-
triaminopyrimidine partial structure have been reported (see
Figure S1a for the structural formula).31,43 These nucleotides
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Figure 2. continued
butanedione, and analyzed for the fluorescent DMPT (9). Also shown
are the chromatograms for the DMPT standard, and for control
reactions lacking GTP, SAM, dithionite, or MoaA. A small amount of
DMPT (∼10% of the complete condition) was formed in the GTP
negative control due to the copurification of GTP with MoaA. (c)
Time course of 3′,8-cH2GTP formation determined after conversion
to DMPT (filled circles) or compound Z (open circle). The conditions
for the MoaA reaction and the derivatization of the product to DMPT
are identical to (b). The amount of compound Z at 60 min is based on
the quantitation of the HPLC peak in (a). Each point is an average of
three replicates, and the error bars are calculated on the basis of the
standard deviation.
have been characterized by derivatization of a hydrolytically
released 6-hydroxy-2,4,5-triaminopyrimidine to the highly
fluorescent DMPT (9; see Figure S1b for the chemical
equation for the derivatization). Schindelin et al. briefly
mentioned in their publication26 that the MoaA reaction
product can be converted to DMPT, although no data were
presented. We thus attempted a similar derivatization to
investigate whether the MoaA product contains an acid-labile 6-
hydroxy-2,4,5-triaminopyrimidine partial structure. For this
purpose, enzyme activity assays were performed with MoaA
(5 μM), GTP (1 mM), SAM (1 mM), and sodium dithionite (1
mM) under anaerobic conditions in the absence of MoaC. The
reaction was stopped by the addition of hydrochloric acid, and
the resulting solution was incubated at 95 °C to facilitate
hydrolysis, followed by incubation with 2,3-butanedione at pH
8. Figure 2b shows the HPLC analysis of this reaction, which
reveals the formation of DMPT. Omission of any of the
reaction components resulted in significant decrease of DMPT.
The time course assay (Figure 2c) revealed that increasing
amounts of DMPT were formed with prolonged incubation
time (Figure 2c). The concentration of DMPT observed in this
Figure 3. (a) Stability of 3′,8-cH2GTP under different conditions.
Solution containing 3′,8-cH2GTP was incubated for 10−180 min
under specified conditions. At each time point, an aliquot was removed
and 3′,8-cH2GTP was quantified by HPLC after its conversion to
compound Z. Each point is an average of three replicates, and the error
bars are calculated on the basis of the standard deviation. (b) UV−vis
absorption spectrum of 3′,8-cH2GTP (bold trace) (50 μM). The thin
traces are spectra after exposure to air at 22 °C for the specified time.
assay at the 60 min time point (3.8
0.2 μM) was
substoichiometric to that of MoaA (5 μM), and within errors
to the amount of cPMP observed in the stepwise assay (3.6
0.3 μM, compare filled and open circles at 60 min time point in
Figure 2c). Together with the results of the stepwise assays,
these preliminary characterizations prompted us to attempt
isolation of 3′,8-cH2GTP.
lowered the yield. Thus, for the isolation of 3′,8-cH2GTP,
GTP concentration was kept stoichiometric relative to MoaA.
Purification conditions also required careful optimization to
avoid decomposition. Successful purification was achieved by
the use of two anion-exchange columns. A strong anion-
exchange resin (QAE Sepharose) was used as the first step of
purification to process a large volume of sample and to separate
3′,8-cH2GTP and GTP from the other components in the
MoaA assay mixture. A weak anion-exchange resin (DEAE
Sepharose) was used to separate GTP from 3′,8-cH2GTP (see
Figure S3 for the chromatograms). Typically, 10 μmol of 3′,8-
cH2GTP was isolated from 200 mL of enzyme reaction mixture
containing 0.2 mM MoaA, 0.2 mM GTP, 1.0 mM SAM, and 1.0
mM sodium dithionite. The isolated 3′,8-cH2GTP had a unique
absorption feature at 322 nm (ε322nm = 5.1 1.1 mM−1 cm−1,
Figure 3b, bold trace). This absorption feature also exhibited
oxygen sensitivity and diminished upon exposure to air (Figure
3b, thin traces), consistent with the oxygen sensitivity of 3′,8-
cH2GTP observed by the biochemical characterization (Figure
3a).
Isolation of 3′,8-cH2GTP. The isolation of 3′,8-cH2GTP
was a significant challenge primarily due to its limited stability.
As shown in Figure 3a, decomposition of 3′,8-cH2GTP was
observed under aerobic conditions or under acidic pH. This
limited stability required all the purifications to be carried out in
an anaerobic glovebox (O2 concentration <0.1 ppm)
maintained at 10 °C using ammonium bicarbonate buffer at
pH 9.1. Under these conditions, no decomposition was
observed at least for 3 h (Figure 3a).
MoaA reaction conditions were chosen to maximize the
production of 3′,8-cH2GTP, as well as the percentage
conversion of GTP to 3′,8-cH2GTP. It was important to
consume as much GTP as possible in the MoaA reaction
because of the close separation between GTP and 3′,8-cH2GTP
during purification with anion-exchange resin. The reaction is
limited by the substoichiometric turnover of MoaA. The
maximum turnover observed after 60 min of incubation at 25
The isolated 3′,8-cH2GTP was also analyzed for the number
of phosphates in the molecule, as its close migration with GTP
on the anion-exchange resin suggested the presence of a
triphosphate group. The use of a colorimetric phosphate
°C was 0.5
0.2 when stoichiometric or excess amount of
GTP relative to MoaA was used (Figure 2c). The use of
substoichiometric amount of GTP to MoaA significantly
assay39 after a phosphatase treatment revealed 2.93
0.03
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1
1
Figure 4. NMR structural characterization of 3′,8-cH2GTP. (a) H NMR (600 MHz, D2O) and (b) H-decoupled 13C NMR (200 MHz, D2O)
spectra of 3′,8-cH2GTP and [U-13C10, 15N5]3′,8-cH2GTP, respectively. No signal associated with 3′,8-cH2GTP was observed outside the shown
chemical shift range. The intensity of the H-1′ signal in (a) was decreased due to the water suppression at 4.95 ppm. The insets in (b) shows
magnified view of the signals for C-3′, C-4′ and C-8. (c) 13C−13C COSY spectrum of [U-13C10,15N5]3′,8-cH2GTP. The assignments for the observed
correlations are indicated. The chemical shift region for C-1′ through C-5′ and C-8 is shown. The spectrum with full chemical shift range is available
1
1
in Figure S6. (d) 13C−13C COSY (bold), and selected H−13C, and H−15N HMBC (single-headed arrows) correlations, and NOE (a double-
headed arrow). A complete list of the observed HMBC correlations is available in Table 1. P designates a phosphate group.
phosphate groups per molecule. This result is consistent with
3′,8-cH2GTP being a triphosphate compound.
cH2GTP rather than contaminating GTP. To distinguish 3′,8-
cH2GTP from GTP, purified 3′,8-cH2GTP was incubated with
MoaC and analyzed by MS, which exhibited no MS signal at m/
z = 521.984, and instead exhibited a signal at m/z = 362.052
0.001 [M−H]− (Figure S4b) corresponding to a hydrate form
of cPMP (m/z [M−H]− calcd for C10H13N5O8P, 362.051).
These findings suggest that the observed MS signal was indeed
associated with the intact 3′,8-cH2GTP, but not contaminating
GTP.
Molecular Weight Determination of 3′,8-cH2GTP. An
ESI-TOF-MS analysis of isolated 3′,8-cH2GTP revealed m/z =
521.984
0.001 [M−H]− (Figure S4a), consistent with the
molecular formula of C10H15N5O14P3 (calcd m/z = 521.983
[M−H]−). The observed mass was also consistent with GTP,
which migrates close to 3′,8-cH2GTP during the anion-
exchange chromatographies (see Figure S3), and occasionally
contaminated 3′,8-cH2GTP. Thus, it was important to verify
that the observed signal was indeed associated with 3′,8-
Structural Characterization of 3′,8-cH2GTP by NMR
Spectroscopy. Further structural characterization of 3′,8-
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Table 1. Summary of NMR Data for 3′,8-cH2GTP
NMR
COSY
HMBC
1H−13C
no.
13C, δ (ppm) (m, JCC(Hz))
1H δ (ppm) (m, JHH(Hz))
1H−1H
13C−13C
1H−15N
NOESY
2
153.6 (s)
4
156.1 (d, 83.1)
110.8 (t, 76.0)
160.0 (d, 68.0)
75.6 (d, 42.5)
90.8 (d, 40.0)
73.0 (t, 40.0)
80.0 (q, 39.9)
75.9 (t, 42.0)
63.7 (d, 44.9)
C-5
5
C-4, C-6
C-5
6
H-8
8
5.20 (s)
5.02 (s)
4.14 (s)
C-3′
C-2′
C-1′, C-3′
C-8, C-2′, C-4′
C-3′, C-5′
C-4′
H-1′, H-4′
H-2′, H-8
H-8
N-9
N-9
H-5′a
1′
2′
3′
4′
5′a
5′b
H-1′, H-2′, H-4′
H-8, H-1′, H-2′, H-5′
4.48 (dd, 3.9, 7.3)
H-5′a, H-5′b
H-4′, H-5′b
H-4′, H-5′a
a
4.25 (dt, 11.3, 6.2)
H-8
a
4.39 (dt, 11.3, 5.3)
a
2
1
3
3
The doublet feature is derived from a J H− H to the geminal H, and the triplet feature is from a J H− H to H-4′ and a J
phosphate group.
to 31P of the
1
1
1
1
31P−1H
cH2GTP was carried out using NMR spectroscopy. Figure 4a
shows the 1H NMR spectrum of purified 3′,8-cH2GTP with six
signals between 4.1 and 5.3 ppm. To confirm that these signals
are associated with 3′,8-cH2GTP, the observed 1H NMR signals
were quantified by comparing the integrals of the observed
signals with that of a known concentration of maleic acid as an
internal standard. The concentration of 0.59 0.14 mM was
determined by this analysis, which agreed well with the
multiplicity of the 13C NMR signal (Figure 4b), and the
13C−13C COSY correlation (Figure 4c) suggested that C-8 is a
methine primary carbon. The 1H−13C HMBC correlations
from H-8 to C-1′, and from H-1′ to C-8 (Figure 4d), suggested
a connection between C-8 and C-1′ through a heteroatom. The
1H−15N HMBC correlations to the same N from H-1′ and
from H-8 (Figure 4d) suggested that the bridging heteroatom is
N. The second heteroatom on C-8 would be either N or O.
The reported chemical shift values for methine primary carbons
covalently bound to an N and an O are 84−97 ppm,31,47−49
whereas those with two N’s are 71−86 ppm.48,50−53 Thus, the
chemical shift observed for C-8 (75.6 ppm) is most consistent
with the presence of the second N on C-8.
The connections of C-1′ and C-8 to nitrogen atoms
suggested the connections to 6-hydroxy-2,4,5-triaminopyrimi-
dine because all nitrogen atoms are associated with the 6-
hydroxy-2,4,5-triaminopyrimidine based on the molecular
formula determined by MS. The connection to 6-hydroxy-
2,4,5-triaminopyrimidine is supported by the observation of a
weak 1H−13C HMBC correlation from H-8 to C-6 (Figures 4d
and S9). While several different orientations for 6-hydroxy-
2,4,5-triaminopyrimidine are consistent with the NMR data
(see Figure S11 for possible structures), the nitrogen bridging
C-8 and C-1′ is likely N-9, considering that the previous
isotope tracer experiments indicated that the N9−C1′ bond is
maintained during the transformation of GTP into cPMP.7,17
The second N on C-8 was assigned as N-7, considering that
C8−N7 bond is present in GTP, and there is no evidence
suggesting that the other Ns are involved in the transformation
of GTP to cPMP. On the basis of these analyses, we propose
the orientation of the 6-hydroxy-2,4,5-triaminopyrimidine
connection as shown in Figure 4d.
concentration (0.67
0.03 mM) biochemically determined
after its conversion to compound Z.
The 13C NMR spectrum (Figure 4b) was recorded on 3′,8-
cH2GTP uniformly labeled with 13C and 15N, prepared from
[U-13C10,15N5]GTP. Four carbon signals were found at 111,
154, 156, and 160 ppm, suggesting the presence of four sp2
carbons. The major multiplicities of the 13C NMR signals
1
13C−13
C
(Figure 4b) are derived from J
coupling. The analysis of
the multiplicities (Figure 4b and Table 1) in combination with
the 13C−13C COSY correlations (Figure S6; all 2D NMR
spectra are available in the Supporting Information) established
the connectivities among the signals at 111, 156, and 160 ppm.
Together with the comparison with the chemical shifts reported
for various purines and pyrimidines (Table S1),44,45 we
assigned the four sp2 carbon signals to the carbons in the 6-
hydroxy-2,4,5-triaminopyrimidine moiety of 3′,8-cH2GTP.
The remaining 13C NMR signals were found between 63.7
and 90.8 ppm with observed 1J
13C−13
C coupling constants of ∼40
Hz (Figure 4b and Table 1), suggesting that they are all sp3
carbons.46 The analysis of the multiplicities in combination
with the 13C−13C COSY correlations (Figure 4c) unambigu-
ously determined the C−C connectivities as shown in Figure
4d. The most notable is the presence of C-3′ as a tertiary
carbon that appeared as a quartet signal in the 13C NMR
spectrum (see the inset of Figure 4b). The connectivity of C-3′
to C-8 in addition to C-2′ and C-4′ was established by the
13C−13C COSY correlations (Figure 4c,d). This feature
distinguishes this molecule from GTP, cPMP, or molecules
that have been previously proposed as MoaA products, 2-
amino-5-formylamino-6-ribofuranosylamino-4-pyrimidinone
triphosphate (6), and pyranopterin triphosphate (7),29,30
because none of these molecules has a tertiary carbon. This
analysis suggested that during the MoaA reaction, the C3′−C8
bond is formed, but the pyranopterin structure is not yet
established.
The structures of the remaining part of the molecule were
based on the following analysis. The furanose ring was based on
1
the H−13C HMBC correlations from H-1′ to C-4′ (Figure
4d). The position of the triphosphate group was based on the
3JHP = 5−6 Hz observed for the 1H NMR signals for H-5′a and
H-5′b (Figure 4a and Table 1), which are comparable to those
reported for various nucleotides.54,55 The hydroxyl groups at
the 2′ and 3′ positions were based on the NMR chemical shifts
observed for H-2′, C-2′, and C-3′. The stereochemistry at C-1′,
C-2′, C-3′, and C-4′ is based on GTP and cPMP.56 The
absence of an apparent J coupling between H-1′ and H-2′
(3JH1′H2′ < 4 Hz) is consistent, based on the Karplus relations,57
Further NMR analysis provided a basis for the complex
1
multi-ring structure around C-8. H−13C HMQC (Figure S7),
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Table 2. Steady-State Kinetic Parameters for MoaA, MoaC, and MOCS1B
enzyme
MoaA
substrate
GTP
Km (μM)
kcat (min−1
)
kcat/Km (μM−1 min−1
)
1.4 0.2
4.1 1.3
<0.060
0.045 0.003
0.043 0.004
0.17 0.026
0.092 0.020
0.032 0.012
0.011 0.003
>2.8
SAM
MoaC
3′,8-cH2GTP
3′,8-cH2GTP
a
MOCS1B
0.79 0.24
0.12 0.08
a
Purified MOCS1B was a complex of a full-length peptide (26 kDa) with two N-terminally truncated peptides (18 and 16 kDa). See Figure S14 for
details.
with the calculated dihedral angle of 59° between H1′−C1′−
C2′−H2′ (see Figure S12 for the energy-minimized con-
formation of 3′,8-cH2GTP). The stereochemistry at C-8 is
based on NOE observed between H-8 and H-5′a (Figure 4d, a
double-headed arrow).
On the basis of all the analyses described above, we propose
the structure of the isolated molecule as (8S)-3′,8-cyclo-7,8-
dihydroguanosine 5′-triphosphate (3′,8-cH2GTP, Figure 4d).
The proposed structure is consistent with all the NMR data
(Table 1), as well as MS, phosphate assay, and chemical
derivatization to DMPT described above.
characterization. The HPLC analysis of the product of the
MOCS1B activity assay with 3′,8-cH2GTP as the substrate
revealed that cPMP was formed with the kinetic parameters of
Km = 0.79 0.24 μM and kcat = 0.092 0.020 min−1, which are
somewhat less efficient than but comparable to Km and kcat
determined for MoaC (Table 2). While these kinetic
parameters could be affected by the observed truncation at
the N-terminus and/or the lack of the MOCS1A domain, the
results show that 3′,8-cH2GTP is a substrate for MOCS1B, and
thus suggest that 3′,8-cH2GTP may be a biosynthetic
intermediate in human Moco biosynthesis as well.
Relevance of 3′,8-cH2GTP to Moco Biosynthesis. The
physiological relevance of 3′,8-cH2GTP as an intermediate of
Moco biosynthesis was further investigated by steady-state
kinetics of the MoaA- and MoaC-catalyzed reactions. The
kinetic parameters for the MoaC-catalyzed conversion of 3′,8-
cH2GTP to cPMP were determined using purified 3′,8-
cH2GTP as the substrate. The kinetic parameters for the
MoaA-catalyzed reaction were determined by a coupled assay in
the presence of a 10-fold excess of MoaC to ensure efficient
conversion of 3′,8-cH2GTP to cPMP. In both assays, cPMP was
converted to compound Z and quantified by HPLC. The
results are summarized in Table 2. The kcat of MoaC was ∼4-
fold greater than that of MoaA determined by this method, and
the Km of MoaC for 3′,8-cH2GTP was <0.06 μM, the lower
limit of detection. Km of MoaA for GTP was found to be 1.4
0.2 μM, which may be compared to the previously reported
Stoichiometry of the MoaA Reaction and the Position
of H-Atom Abstraction. Since MoaA is a member of the
radical SAM superfamily, the conversion of GTP to 3′,8-
cH2GTP is likely initiated by a radical formation at C-3′ by 5′-
dA• formed by reductive cleavage of SAM, followed by the
attack of C-3′ to C-8. In the reactions catalyzed by radical SAM
enzymes, SAM may be consumed as a co-substrate, or
regenerated and used as a co-catalyst.23 Since the conversion
of GTP into 3′,8-cH2GTP could take place either of the two
mechanisms, we quantified the amount of 5′-dA and cPMP
formed in the MoaA/MoaC coupled assay. A 2-fold excess of
MoaC was used relative to MoaA to ensure complete
conversion of 3′,8-cH2GTP to cPMP. HPLC quantitation of
5′-dA suggested that, under this condition, stoichiometric
amounts of 5′-dA and 3′,8-cH2GTP were formed (Figure 5a).
The determination of the stoichiometry of SAM cleavage for
radical SAM enzymes has been frequently obscured by abortive
production of 5′-dA.58 While our data are consistent with
stoichiometric formation of 5′-dA, it is important to show that
the observed 5′-dA is indeed the product of H-atom abstraction
from GTP, but not a product of abortive SAM cleavage. Thus,
we investigated the transfer of a deuterium atom on GTP into
5′-dA. Based on the structure of 3′,8-cH2GTP, the reaction
most likely proceeds through H-abstraction from the 3′
position. Thus, [3′-2H]GTP was chemoenzymatically prepared
from [3-2H]ribose, and used to investigate a deuterium transfer
from [3′-2H]GTP to 5′-dA. When [3′-2H]GTP was used as a
substrate, a small but significant kinetic isotope effect of VH/VD
= 1.28 0.05 was observed (Figure 5b), which is comparable
to a value previously reported for another radical SAM enzyme,
BtrN (VH/VD = 1.3 0.1).59
binding constant of 0.29
0.11 μM determined by an
equilibrium dialysis.26 These observations support the con-
clusion that 3′,8-cH2GTP is likely the physiological substrate of
MoaC, and the biosynthetic intermediate between GTP and
cPMP during Moco biosynthesis.
The relevance of 3′,8-cH2GTP as an intermediate in human
Moco biosynthesis was investigated by the characterization of a
human homologue of MoaC. Previous gene complementation
studies suggested that the human proteins, MOCS1A and
MOCS1B, have functions that correspond to those of MoaA
and MoaC, respectively.37 In human cells, MOCS1B is
expressed as an N-terminal fusion with a catalytically inactive
MOCS1A.20,37 Since we have not been successful in obtaining
the fusion protein, the stand-alone MOCS1B domain was
expressed as an N-terminal His-tag protein, and purified by Ni-
affinity chromatography. SDS-PAGE and MS analysis of the
purified protein revealed that MOCS1B (26 kDa) was co-
purified with two other shorter MOCS1B peptides (16 and 18
kDa) truncated at the N-terminus (see Figure S14 for details of
characterization). The ratio of the truncated MOCS1B to the
full length MOCS1B was not altered by the use of protease
inhibitor cocktails or further purification by anion-exchange,
size exclusion, and phenyl sepharose column chromatography.
Because the truncation occurred only in the poorly conserved
N-terminal region of MOCS1B (see Figure S14c for a multiple
sequence alignment), we used this MOCS1B for further
To investigate the H-3′ transfer from GTP to 5′-dA, 5′-dA
was isolated from a large scale MoaA/MoaC coupled reaction
mixture, where [3′-2H]GTP (98 μM) was incubated with
MoaA (50 μM), MoaC (100 μM), SAM (1 mM), and sodium
dithionite (1 mM) at 25 °C for 60 min. HPLC analysis of this
assay solution revealed formation of 48 2 μM of 5′-dA and 47
2 μM of cPMP. 5′-dA was isolated using reverse-phase silica
gel column chromatography and HPLC, and characterized by
1
2
MS (Figure 5c), and H and H NMR spectroscopies (Figure
5d). These analysis revealed incorporation of a single
deuterium atom in the 5′-methyl group of 5′-dA, consistent
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with a recent observation by Mehta et al.30 Under the
conditions we investigated, ∼30% of 5′-dA was formed with
no deuterium incorporation (Figure 5c). We attribute this
mainly to the incomplete labeling of [3′-2H]GTP (94 3 atom
2
% H) and the presence of non-labeled GTP copurified with
MoaA. The amount of co-purified GTP was determined as 16
1% that of MoaA by HPLC after its release from MoaA by
acid denaturing. Together with the ∼50% conversion of
[3′-2H]GTP to cPMP, these observations suggest that majority
of the non-labeled 5′-dA formed in this reaction was originated
from the non-labeled GTP in the sample. Thus, our observation
is consistent with a mechanism in which MoaA catalyzes H-
abstraction from the 3′ position of GTP by stoichiometric
consumption of SAM.
DISCUSSION
■
This report describes the isolation and structural character-
ization of 3′,8-cH2GTP, a missing intermediate in Moco
biosynthesis. Our stepwise assay indicated that there is a small
molecule that is produced by MoaA in the presence of GTP,
SAM, and dithionite, and that may serve as a substrate of
MoaC. This compound was isolated from the in vitro MoaA
assay mixture using two sequential anion-exchange column
chromatographies under strict anaerobic conditions, and
structurally characterized by chemical derivatization, MS and
NMR spectroscopy. The physiological relevance of the isolated
3′,8-cH2GTP was investigated by the steady-state kinetic
analysis of MoaC or its human homologue, MOCS1B. In
these analyses, both enzymes catalyze the conversion of 3′,8-
cH2GTP to cPMP with high specificities (Km values of <0.060
and 0.79 μM, respectively). Although the observed turnover
rates for MoaA (kcat = 0.053 min−1) and MoaC (kcat = 0.17
min−1) were slow, they are typical for enzymes in cofactor
biosynthesis,60,61 including other steps of Moco biosynthesis,62
which may be attributed to the small amount of cofactors
required in the cells. Thus, our data presented here suggest that
3′,8-cH2GTP may represent the physiologically relevant
intermediate of Moco biosynthesis.
The MoaA reaction product has been described previously.
Schindelin and Hanzelmann26 first stated in their publication
̈
that in the reaction of MoaA with SAM and GTP without
MoaC they observed the formation of small molecule that may
be converted to DMPT. Unfortunately, no data were presented.
Our analysis in this report provides strong evidence that MoaA
product indeed has an acid-labile 6-hydroxy-2,4,5-triaminopyr-
imidine moiety that may be converted to DMPT. More
recently, Mehta et al. reported the observation of a molecule
with a light absorption feature at 320 nm in the LCMS analysis
of the reaction mixture of MoaA incubated with GTP, SAM,
and dithionite. The observation of a mass signal at m/z = 524
[M+H]+ at the same retention time led the authors to propose
this molecule as pyranopterin triphosphate (7, Figure 1c).
While this conclusion is distinct from ours, the reported light
absorption feature and the mass signal are also consistent with
3′,8-cH2GTP. In addition, Mehta et al. mentioned that the
observed molecule was oxygen sensitive, which is also a
characteristic of 3′,8-cH2GTP. On the other hand, our NMR
data are inconsistent with pyranopterin triphosphate. Thus, we
conclude that the previously described putative MoaA
products26,30 were indeed 3′,8-cH2GTP.
Figure 5. Mechanistic studies on the MoaA-catalyzed H-atom
abstraction. (a) Stoichiometry of formation of 5′-dA (open circles)
and cPMP (filled circles) in the MoaA/MoaC coupled assay. MoaA
(20 μM) was incubated with GTP (1 mM), SAM (1 mM) and sodium
dithionite (1 mM) in the presence of MoaC (40 μM) at 25 °C. cPMP
was quantified by HPLC after its conversion to compound Z. Each
point is an average of 3−6 replicates, and the error bars are calculated
on the basis of the standard deviation. (b) Rate of cPMP formation
from GTP or [3′-2H]GTP by MoaA and MoaC. MoaA (0.5 μM) was
incubated with GTP or [3′-2H]GTP (0.1 mM) in the presence of
sodium dithionite (1 mM), SAM (1 mM) and MoaC (5 μM) at 25 °C.
cPMP was quantified by HPLC after its conversion to compound Z.
Each point is an average of four replicates, and the error bars are
calculated on the basis of the standard deviation. (c) ESI-TOF-MS and
(d) 1H NMR (600 MHz in D2O) and 2H NMR (76.75 MHz in H2O)
spectra of 5′-dA isolated from the reaction in the presence of MoaA
(50 μM), MoaC (100 μM), SAM (1 mM), [3′-2H]GTP (98 μM, 94
2
3% atom H), and sodium dithionite (1 mM). The signal indicated
with * is non-deuterated 5′-dA (m/z [M+H]+ calcd for C10H14N5O3
252.110, found 252.111), presumably derived from non-labeled GTP
(see main text).
It is noteworthy that Mehta et al. also considered 3′,8-
cH2GTP as an intermediate of cPMP biosynthesis, but
suggested to exist only transiently during the MoaA-catalyzed
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Figure 6. Proposed mechanism for the MoaA-catalyzed conversion of GTP to 3′,8-cH2GTP.
conversion of GTP to pyranopterin triphosphate.30 This
proposal appears to be based mostly on their observation of
the deuterium transfer from the 3′ position of GTP to 5′-dA,
and their proposal for pyranopterin triphosphate as a MoaA
product, which we now believe to be a mis-assignment. While
their deuterium tracer experiments support the formation of a
radical at C-3′, prior to our work, to our knowledge, there was
no precedent for the reaction between a C-3′ centered radical
and C-8 in purine nucleosides or nucleotides. In addition, the
radical formation at C-3′ could also be consistent with the
previous proposals with 6 as an intermediate (Figure 1b)29,33 if
the H-3′ abstraction was to take place after the formation of 6.
There are precedents for formyl group transfer reactions by
radical mechanisms.63 Our isolation and characterization of
3′,8-cH2GTP as a MoaA product provides strong evidence for
the function of MoaA to catalyze the conversion of GTP to
3′,8-cH2GTP. As 3′,8-cH2GTP can be chemically derivatized to
DMPT, our model is also consistent with the observations by
by abortive cleavage of SAM, where SAM is reductively cleaved
to methionine and 5′-dA regardless of product formation. The
abortive SAM cleavage has been reported for many radical SAM
enzymes to various degrees.58,70−74 A notable example is the
recent report on QueE by McCarty et al.70 QueE catalyzes a
complex rearrangement of 7-carboxy-7-deazaguanine to 6-
carboxy-5,6,7,8-tetrahydropterin during deazapurine biosyn-
thesis. Although SAM is used catalytically in this reaction,
detectable amount of 5′-dA was formed and contained
deuterium atoms that originated from substrate. Thus, although
previous studies indicated a transfer of a deuterium atom at the
3′ position of GTP to 5′-dA,30 the lack of knowledge about the
stoichiometry of the reaction left significant ambiguity about
how SAM is used in the MoaA reaction. Our observation of the
stoichiometric formation of 5′-dA and cPMP in the MoaA/
MoaC coupled assay, in combination with the demonstration of
the stoichiometric transfer of a deuterium atom at the 3′
position of GTP to the methyl group of 5′-dA, is consistent
with the stoichiometric consumption of SAM and H-atom
abstraction from the 3′ position.
On the basis of the observations described above and our
structural characterization of 3′,8-cH2GTP, in combination with
chemical precedents and the reported structures of MoaA, we
propose a mechanism for the MoaA-catalyzed reaction (Figure
6). Our deuterium tracer experiments and those reported by
Mehta et al.30 suggested that the reaction is initiated by the
abstraction of H-3′ by 5′-dA•, generated by the reductive
cleavage of SAM using the N-terminal 4Fe-4S cluster as a
reductant. The resulting C-3′ radical (10) then attacks C-8 and
generates a 3′,8-cycloGTP aminyl radical intermediate 11,
which is then converted to 3′,8-cH2GTP. While the aminyl
radical 11 could be reduced by a re-abstraction of H-atom from
5′-dA, our observation of the stoichiometric formation of 5′-dA
in the MoaA/MoaC coupled assay relative to cPMP disfavors
such mechanism. Thus, another electron source is required.
One possible reductant is the C-terminal [4Fe-4S] cluster that
binds GTP.26,27 A reduction of 5′,8-cyclo-deoxyadenosine
aminyl radical by reduced methylviologen (E° = −0.45 V)75
has been reported. Thus, considering the reported redox
potentials of related [4Fe-4S] clusters (E° = −0.4 to −0.6
V),76,77 the C-terminal [4Fe-4S] cluster may carry out the
reduction of the aminyl radical. The redox role of the C-
terminal [4Fe-4S] cluster may be probed by EPR spectrosco-
py36 and is currently being investigated in our laboratory.
The reduction of the aminyl radical 11 may also be facilitated
by the three conserved Arg residues, R17, R266, and R268
(numbering based on S. aureus MoaA), which are 3.2−4.6 Å
from N-7 of GTP in the crystal structure.26 Mutations in these
residues have been found in Moco-deficient patients.78 MoaA
variants with mutations in these residues still bind GTP to a
various degree, but do not produce cPMP when assayed with
Schindelin and Hanzelmann.26
̈
Our identification of 3′,8-cH2GTP as the product of MoaA as
well as the substrate of MoaC is a sharp contrast to the previous
consensus in the field, where MoaA was considered largely
responsible for the formation of the pyranopterin ring, and a
relatively minor role was considered for MoaC.29,30 Our
observations suggest that MoaC plays a major role in the
formation of pyranopterin ring. The reactions catalyzed by
MoaA and MoaC may be compared to the conversion of GTP
into 7,8-dihydroneopterin triphosphate by GTP cyclohydrolase
I in folate biosynthesis (see Figure S1a). The first step of the
GTP cyclohydrolases I reaction is the hydrolysis of the
imidazole moiety of the guanine base using a water activated
on Zn2+ in the active site.31,64 This hydrolysis triggers the
ensuing complex Amadori rearrangement18,65−67 that takes
place in the same active site.68,69 Our results suggest that Moco
biosynthesis is also initiated by modification of GTP at the C-8
position, but to a distinct molecule, 3′,8-cH2GTP. In contrast
to GTP cyclohydrolases I, MoaA is incapable of catalyzing the
rearrangement reactions, presumably due to the lack of
appropriate amino acid residues in the active-site. Instead,
3′,8-cH2GTP is transferred to MoaC where a complex
rearrangement reaction proceeds. In the following paragraphs,
we will discuss the mechanisms of the reactions catalyzed by
each of these two enzymes.
The observation of 3′,8-cH2GTP formed from GTP by a
radical SAM enzyme, MoaA, indicated to us that the reaction
would proceed through a H-atom abstraction from the 3′
position. This is consistent with the deuterium tracer
experiments by Mehta et al.30 In reactions catalyzed by radical
SAM enzymes, SAM may be used catalytically or stoichio-
metrically. However, the demonstration of the stoichiometry
for radical SAM-catalyzed reactions is frequently complicated
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MoaC.26 These Arg residues may be assisting the aminyl radical
reduction by electrostatic stabilization of the anionic charge on
the base, or by providing a H+. Controlled movements of
protons and electrons are often critical for redox reactions in
enzyme catalysis.79
Another important feature of the active-site of MoaA is the
presence of conserved hydrophobic amino acid residues, V167,
I194, and I253, surrounding 2′-OH and 3′-OH of GTP (Figure
7).26 No amino acid residues are in H-bonding interaction
conversion of GTP into cPMP. Thus, our finding that 3′,8-
cH2GTP may be the physiological substrate provides a means
to study the mechanism of the complex rearrangement reaction
catalyzed by MoaC.
The structures of MoaC32−35,83 or previous in vitro
characterization of MoaC in the presence of MoaA9 did not
reveal any complex cofactor or metals in MoaC. Thus, the
MoaC reaction appears to take place via general acid/base
catalysis using amino acid side chain of MoaC. Our
characterization of 3′,8-cH2GTP suggested susceptibility of
this molecule to acid hydrolysis. Thus, the transformation of
GTP into 3′,8-cH2GTP by MoaA may be sufficient to make this
molecule susceptible to complex rearrangement by MoaC
presumably using general acid/base catalysts. While several
different mechanisms may be possible, Figure 8 shows a
Figure 7. Model of the MoaA active site. SAM was modeled into the
X-ray crystal structure of MoaA in complex with GTP26 (PDB ID:
2FB3) based on the structural alignment with the X-ray crystal
structure of MoaA in complex with SAM9 (PDB ID: 1TV8). Oxygens
are shown in red, nitrogens in blue, sulfurs in yellow, phosphorus in
orange, and irons in brown.
Figure 8. Possible mechanism of the MoaC-catalyzed conversion of
3′,8-cH2GTP to cPMP. P designates a phosphate group.
distance from 2′-OH and 3′-OH, which is inconsistent with the
earlier proposal where MoaA catalyzes complex rearrangement
reactions that require general acid/base catalysis at 3′-OH.30
On the other hand, the structural feature of MoaA active site is
consistent with our proposal for the function of MoaA to
catalyze the conversion of GTP to 3′,8-cH2GTP, in which no
general acid/base catalysis is required.
possible mechanism for the conversion of 3′,8-cH2GTP to
cPMP by MoaC most consistent with the currently available
chemical and enzymological precedents. The reaction is likely
initiated by hydrolysis of the constrained pyrrolidine and
imidazoline rings of 3′,8-cH2GTP to an aldehyde 12, based on
the susceptibility of 3′,8-cH2GTP to acid hydrolysis during
derivatization to DMPT (Figure 2b). Acid hydrolysis of various
aminal molecules has been well documented.84 The aldehyde
12 is then converted to a hexose 13. The mechanism of this
reaction could be either a retroaldol−aldol rearrangement
initiated by the deprotonation at 2′-OH, or an α-ketol
rearrangement initiated by the deprotonation at 3′-OH. A
similar rearrangement reaction has been reported for 1-deoxy-
D-xylulose-5-phosphate reductoisomerase.85 The hexose 13 is
then converted to cPMP by formation of the pterin ring and
the cyclic phosphate. Considering that MoaC binds nucleoside
triphosphate stronger than di- and monophosphate,33 the cyclic
phosphate formation may be the last or close to the last step of
the catalysis. Studies to further narrow the possible mechanisms
are currently underway.
The hydrophobic environment around 2′-OH and 3′-OH of
GTP may also be important for directing the reaction of C-3′
centered radical with C-8 by preventing side reactions. To our
knowledge, the reaction of C-3′ radical with C-8 of purine
nucleoside/nucleotide is unprecedented.28,42,80 Radical forma-
tion at C-3′ in DNA and RNA results in a formation of 3′-keto-
2′-deoxyribonucleotides or a C3′−C4′ bond cleavage by
complex mechanisms.80 Reactions of the C-3′ nucleotide
radical in enzymes have been extensively studied for
ribonucleotide reductase (RNR),81,82 where the radical
formation on C-3′ facilitates dissociation of 2′-OH. Mechanistic
studies on RNR and related synthetic model systems suggest
that the dissociation of 2′-OH requires general acid/base
catalysts at 2′-OH and 3′-OH.82 The difference in the active-
site structures of MoaA and RNR that both generate a radical at
C-3′, but produce distinct products, may indicate the
mechanism by which enzymes control radical reactions. The
roles of the MoaA active site amino acid residues are currently
under investigation.
CONCLUSION
■
This work describes the isolation and characterization of 3′,8-
cH2GTP, an intermediate in Moco biosynthesis, which had
previously eluded structural characterization. Our finding
defines the reactions catalyzed by MoaA and MoaC and
provides insights into their catalytic mechanisms. In contrast to
the previous notion in the field, our results suggests a complex
mechanism for the MoaC-catalyzed reaction.
The following complex rearrangement reactions are catalyzed
by MoaC. While MoaC was previously known to be essential
for the biosynthesis of cPMP,7,9,20,21 the lack of understanding
about the structure of the substrate of MoaC precluded
identification of the exact role of this enzyme during the
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(13) Nichols, J. D.; Rajagopalan, K. V. J. Biol. Chem. 2005, 280,
7817−7822.
ASSOCIATED CONTENT
* Supporting Information
■
S
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Detailed protocol for expression and purification of S. aureus
MoaA and MoaC; 6-hydroxy-2,4,5-triaminopyrimidine nucleo-
tides in the pterin and flavin biosynthesis; SDS-PAGE and
UV−vis spectra of MoaA and MoaC; chromatograms of 3′,8-
cH2GTP purification; MS characterization of 3′,8-cH2GTP;
energy-minimized conformation of the nucleoside moiety of
3′,8-cH2GTP; 2D NMR spectra of 3′,8-cH2GTP; steady-state
kinetic analysis of MoaA, MoaC, and MOCS1B; MS character-
ization of MOCS1B; MoaA assay with [3′-2H]GTP; 13C NMR
summary for purine and pyrimidine bases. This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
ken.yoko@duke.edu
■
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Miller, N. E. Nucleic Acids Res. 2001, 29, 1097−1106.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(25) Vey, J. L.; Drennan, C. L. Chem. Rev. 2011, 111, 2487−2506.
This work was supported by start-up funds from the Duke
University Medical Center (to K.Y.), and in part by National
Institutes of Health (NIH) Grant P30-CA014236 (to A.A.R.).
We acknowledge the Duke University NMR Spectroscopy
Center shared resource for spectrometer time. Instrumentation
in the Center was purchased with support from NIH, NSF,
HHMI, and the North Carolina Biotechnology Center. We
thank George R. Dubay (Duke University, Department of
Chemistry) for assistance on the MS measurements. We
acknowledge the Duke proteomics core facility for the MS
analysis of MOCS1B. We acknowledge J. A. Gerlt (University
of Illinois at Urbana−Champaign) for providing an expression
plasmid for 5-methylthioribose kinase, and Petra Hanzelmann
and Hermann Schindelin (University of Wurzburg) for
providing expression plasmids for the MOCS1B and SUF
proteins. We thank Dr. JoAnne Stubbe (MIT, Departments of
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