A Radical dance in thiamin biosynthesis: Mechanistic analysis of the bacterial hydroxymethylpyrimidine phosphate synthase

Article abstract of DOI:10.1002/anie.201003419

Tricky things with ThiC: Hydroxymethylpyrimidine phosphate (HMP-P) synthase (ThiC) catalyzes one of the most complex rearrangement reactions in primary metabolism. Deuteration experiments show that under reducing conditions, in the presence of aminoimidazole ribonucleotide, the 5-deoxyadenosyl radical generated at the active site of ThiC reacts directly with the substrate and performs two iterative hydrogen atom abstraction events to catalyze this rearrangement (see scheme; SAM=S-adenosylmethionine).

Full text of DOI:10.1002/anie.201003419

Angewandte
Chemie
DOI: 10.1002/anie.201003419
Thiamin Biosynthesis
A “Radical Dance” in Thiamin Biosynthesis: Mechanistic Analysis of
the Bacterial Hydroxymethylpyrimidine Phosphate Synthase**
Abhishek Chatterjee, Amrita B. Hazra, Sameh Abdelwahed, David G. Hilmey, and
Tadhg P. Begley*
Thiamin pyrophosphate is an important cofactor in all forms
of life, where it plays a central role in the stabilization of the
[
1,2]
acyl carbanion biosynthon.
Its biosynthesis involves sepa-
rate syntheses of the thiazole and the pyrimidine heterocycles,
which are then linked to form the cofactor. Thiamin thiazole
[3–7]
biosynthesis is relatively well-understood.
In prokaryotes,
1
-deoxy-d-xylulose-5-phosphate, cysteine, and glycine or
tyrosine are utilized by five proteins to construct the thiazole
moiety, whereas in Saccharomyces cerevisiae, just one gene
product converts NAD and glycine to thiazole, obtaining
sulfur from a source yet unknown. In contrast, the mecha-
nistic understanding of thiamin pyrimidine (HMP) biosyn-
thesis, in both prokaryotes and eukaryotes, is still at an early
stage. In yeast, a single gene product, THI5p, is implicated in
HMP biosynthesis from PLP (pyridoxal 5’-phosphate) and
histidine, however this reaction has not yet been successfully
reconstituted in vitro. In bacteria and plants HMP-P synthase
Scheme 1. The pyrimidine synthase-catalyzed conversion of AIR (1) to
HMP-P (2; P=phosphate). Reduction of SAM (3) generates the 5-
deoxyadenosyl radical (4), which triggers the rearrangement reaction
by hydrogen atom abstraction from AIR (1). The colors in 1 and 2
show the origin of the atoms of HMP-P in the AIR structure.
(
ThiC) catalyzes the conversion of aminoimidazole ribonu-
cleotide (AIR, 1), an intermediate in the purine nucleotide
[
10]
biosynthesis pathway, to hydroxymethylpyrimidine phos-
recently described,
it is now possible to identify these
[8]
phate (HMP-P, 2). In vivo and in vitro studies on the
reaction products. This identification is essential to under-
stand the mechanism of the ThiC-catalyzed reaction.
Enzymes belonging to the radical SAM superfamily of
proteins initiate catalysis by hydrogen atom abstraction
from the protein or substrate by the reactive 5’-deoxyadenosyl
reaction catalyzed by ThiC, using labeled AIR, have revealed
a
rearrangement reaction of remarkable complexity
[
9]
(
Scheme 1). The ThiC-catalyzed reaction has recently
been reconstituted in a defined biochemical system. Spectro-
scopic, structural, and biochemical studies established this
enzyme as a unique member of the [4Fe-4S] cluster depen-
[
12–14]
radical (4, Scheme 1).
Here we also report the identifi-
cation of the hydrogen atoms abstracted from AIR, initiating
a remarkable “radical dance” leading to the conversion of
AIR (1) to HMP-P (2).
To evaluate the fates of C1’ and C3’, [ C-1’]-AIR and [ C-
3’]-AIR were synthesized. Using these as substrates, reactions
were set up with ThiC, SAM, and dithionite. A SAM-free
control reaction was also performed. After removal of the
protein, HPLC analysis showed approximately 45% conver-
sion of AIR to HMP-P while no conversion was detected in
the control. C NMR analysis of the reaction mixture
generated using AIR labeled on C1 (Figure 1A, B) showed
a singlet at 170 ppm, which was absent in the control.
Addition of sodium formate increased the intensity of this
signal, confirming its assignment as the formate carbon. This
demonstrates that C1’ of AIR is converted to formate. When
[10,11]
dent radical SAM (S-adenosylmethionine) superfamily.
Labeling studies, in vivo and using cell free extract, have
established the origin of all the thiamin pyrimidine carbon
1
3
13
[9]
and nitrogen atoms in the AIR structure (Scheme 1). These
studies relied on the ease of isolation of thiamin and therefore
could only elucidate the fate of atoms incorporated into
HMP-P. The complexity of living cells and cell free extract
made it impossible to identify the fate of C1’ and C3’ of the
AIR ribose (Scheme 1) as these atoms are not incorporated
into thiamin. With the defined ThiC reconstitution system
1
3
[
+]
[+]
[
*] A. Chatterjee, A. B. Hazra, S. Abdelwahed, D. G. Hilmey,
Prof. T. P. Begley
Department of Chemistry, Texas A&M University
College Station, TX 77843 (USA)
1
3
identical experiments were performed with [ C-3’]-AIR, no
1
3
new signal was observed in the C NMR spectrum, even
though significant conversion of AIR to HMP-P (40–50%)
was confirmed by HPLC analysis. This suggests that C3’ is not
released as formate. Attempts were then made to detect
formaldehyde in the reconstitution mixture, directly by
Fax: (+1)979-458-5735
E-mail: begley@chem.tamu.edu
+
[
] These authors contributed equally to this work.
[
**] This research was funded by NIH grant DK44083 and by the Robert
A. Welch Foundation (A-0034)
1
3
[17]
C NMR before
and after removal of the protein and
Generation of formaldehyde during the
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201003419.
[
20]
using Purpald.
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Communications
to the concentration of AIR used in the reaction mixture
Figure 1D). We therefore conclude that the C3’ of AIR is
(
converted to carbon monoxide.
Having established the fates of the C1’ and C3’ atoms of
AIR, we next investigated the role of the 5’-deoxyadenosyl
radical in the reaction. As a member of the radical SAM
superfamily, the 5’-deoxyadenosyl radical, generated by
reduction of SAM, plays an intimate role in triggering the
rearrangement of AIR to HMP-P. This radical may abstract a
hydrogen atom directly from the substrate or, alternatively,
abstract a hydrogen atom from the protein to generate a
[16]
protein-bound radical, which then reacts with the substrate.
When it directly reacts with the substrate, the 5’-deoxyade-
nosyl radical may be used as a co-substrate or as a catalyst, in
which case it is regenerated at the end of the reaction. To
identify which hydrogen atom from AIR is abstracted by the
5’-deoxyadenosyl radical, four deuterium-labeled AIR iso-
topomers and an AIR di-deuterated at the 5’ position of
ribose were synthesized (Figure 2A) and the 5-deoxyadeno-
sine generated from each was purified by HPLC. Incorpo-
ration of a deuterium atom at the 5’ position of 5’-deoxy-
1
adenosine results in a small upfield shift of the 5’ H NMR
Figure 1. Determining the fate of C1’ and C3’ of AIR: A) NMR analysis
1
3
of the pyrimidine synthase reaction mixture using AIR labeled with
C
1
3
at the 1 position of the substrate: 1) C signal for C1’ of AIR (88 ppm)
in the SAM-free control reaction; 2) full reaction mixture showing a
new signal at 171 ppm along with the C1’ signal of unreacted AIR;
3
) addition of 30 mm sodium formate to the reaction mixture enhan-
ces the intensity of the new peak at 171 ppm. B) An expanded view of
the NMR spectra around the formate peak at 171 ppm for samples (2)
and (3). C) UV/Vis spectrum of hemoglobin (green) and carboxyhemo-
globin (black) and the resulting difference spectrum. D) The ThiC-
catalyzed reaction run in the presence of hemoglobin shows that
carboxyhemoglobin production is dependent on the AIR concentration.
ThiC-catalyzed reaction was not detected by either method.
To test for carbon monoxide production, an anaerobic carbon
Figure 2. Hydrogen atom abstraction from AIR occurs at C4’ and at
C5’. A) Deuterium-labeled substrates synthesized for this study.
B) H NMR signal for the 5’-methyl group of 5-deoxyadenosine isolated
[
19]
1
monoxide (CO) trapping assay, which involves a specific
change in the Soret-region absorbance of hemoglobin (Fig-
ure 1C) as a result of its association with CO to form
carboxyhemoglobin, was adapted. With this assay, the for-
mation of CO during the ThiC-catalyzed conversion of AIR
to HMP-P was clearly detected. To confirm this result, the
experiment was performed with different concentrations of
AIR substrate, and the change in the signal was proportional
from the ThiC reactions with the AIR isotopomers. Incorporation of
deuterium in the methyl group for [4-D]-AIR (4D) and [5,5’-D ]-AIR
2
(
5,5’D ) is noted by the presence of the additional, upfield shifted
2
broader doublet. C) Rate of formation of 5’-deoxyadenosine and HMP-
P measured over 5 h reveal a 1:1 product ratio. D) MS analysis of 5’-
deoxyadenosine produced in the early phase of the reaction reveal
predominant monodeuteration (m/z 253) with [4-D]-AIR and [5-D]-AIR
and 50% dideuteration (254 Da) with [UD]-AIR.
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[
15]
1
signal.
Therefore, H NMR analysis of the isolated 5’-
AIR was synthesized from this and subjected to the ThiC-
deoxyadenosine was used to detect deuterium incorporation.
Surprisingly, we observed deuterium incorporation into 5’-
deoxyadenosine when [5,5’-D ]-AIR (5,5’D ) or [4-D]-AIR
catalyzed reaction. The resulting 5’-deoxyadenosine was
analyzed by HPLC-coupled ESI-MS (Figure 2D; UD
sample). The mass spectrum shows the production of [CH3-
5’]-deoxyadenosine, [CH D-5’]-deoxyadenosine and [CHD -
2
2
(4D) was used as the substrate. No deuterium incorporation
2
2
was associated with the 1’, 2’, or 3’-labeled substrates (Fig-
ure 2B).
To determine if the 5’-deoxyadenosyl radical is used as a
co-substrate or as a catalyst, we determined the product ratio
5’]-deoxyadenosine in close to a 1:1:1 ratio. [CH -5’]-deoxy-
adenosine is likely to be produced by the quenching of the
adenosyl radical by buffer components (uncoupled reaction),
3
[CH D-5’]-deoxyadenosine is produced by the abstraction of
2
(
HMP-P:5’-deoxyadenosine) of the ThiC-catalyzed reaction
a single deuterium and a hydrogen from the substrate, and the
over a period of 5 h using [5,5’-D ]-AIR (5,5’D ) and [4-D]-
only way that [CHD -5’]-deoxyadenosine can be produced is
2
2
2
AIR (4D) as substrates. A product ratio of nearly 1:1 was
observed throughout the course of the reactions (Figure 2C).
Flavodoxin–flavodoxin reductase and NADPH were used to
reduce the Fe–S cluster of ThiC, since the use of dithionite as
the reducing agent was associated with high levels of
uncoupled 5’-deoxyadenosine production. Although NMR
analysis was used initially to detect deuterium incorporation
in 5’-deoxyadenosine, it was not suitable for quantitative
analyses. In addition to the signals from monodeuterated and
non-deuterated 5’-deoxyadenosine being convoluted, pro-
longed incubation of the reaction mixture (16 h) was required
to generate enough 5’-deoxyadenosine for its isolation and
characterization by NMR spectroscopy. Enhanced levels of
uncoupled 5’-deoxyadenosine pro-
by the sequential abstraction of two deuterium atoms from
the substrate. The observed 1:1 distribution of mono- vs. bis-
deuterated 5’-deoxyadenosine (m/z 253 and 254, respectively)
is consistent with the deuteration of the substrate (98% at
C5’, 50% at C4’). Since HMP-P and 5’-deoxyadenosine are
formed in a 1:1 ratio, these results confirm that two hydrogen
atoms, one from C4’ the other from C5’ of AIR are
incorporated into a single 5’-deoxyadenosine during the
course of the ThiC-catalyzed reaction.
ThiC catalyzes one of the most complex rearrangement
reactions in primary metabolism. Successful anaerobic purifi-
cation of the ThiC enzyme and its reconstitution in a
chemically defined system has set the stage for the elucidation
duction towards the later part of
the reaction introduces a higher
population of unlabeled 5’-deoxy-
adenosine in a sample prepared in
this manner. Therefore, HPLC-
coupled ESI-MS analysis of the
5’-deoxyadenosine was performed
to allow sensitive product detec-
tion at the earlier points of the
reaction, where uncoupled 5’-
deoxyadenosine production was
minimal, and revealed monodeu-
terated 5’-deoxyadenosine as the
predominant product in both cases
(
Figure 2D). These results dem-
onstrate that the 5’-deoxyadenosyl
radical is used as a co-substrate,
which abstracts two hydrogen
atoms for each HMP-P produced,
rather than as a catalyst. This
suggests that the same adenosyl
radical abstracts hydrogen atoms
from the 5’ and the 4’ positions of
AIR.
To further test this unprece-
dented deoxyadenosyl radical
reactivity, perdeuterated AIR
was synthesized and tested as a
substrate. The synthesis used cata-
lytic H/D exchange to produce
ribose that was fully deuterated
[
17]
(> 98%)
at C2’, C3’, and C5’
and partially deuterated at posi-
tions C4’ (50%) and C1’ (< 1%).
Scheme 2. Mechanistic proposal for the rearrangement catalyzed by ThiC (RC=5’-deoxyadenosyl
radical, R-H=5’-deoxyadenosine).
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8655

Communications
of the mechanism of this novel deep-seated rearrangement.
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16]
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rearrangement reaction and the 5’-deoxyadenosyl radical
carries out two iterative hydrogen atom abstractions. This
suggests that a hydrogen atom abstraction from C4’ or C5’ of
the substrate initiates some reaction steps which ultimately
regenerate the 5’-deoxyadenosyl radical followed by a second
hydrogen atom abstraction leading to completion of the
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9
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the very complex rearrangement involved in conversion of
AIR to HMP-P. A mechanistic proposal consistent with our
observations thus far is outlined in Scheme 2. However,
further experiments to determine the order of these hydrogen
atom abstractions by the 5’-deoxyadenosyl radical and to trap
intermediates on the reaction pathway are necessary to clarify
our mechanistic analysis of this remarkable “radical dance”.
[
[
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4
Received: June 5, 2010
Published online: September 30, 2010
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6
Keywords: carbon monoxide · enzymes · radical rearrangement ·
[20] N. W. Jacobsen, R. G. Dickinson, Anal. Biochem. 1974, 46, 298 –
.
S-adenosylmethionine · thiamin
299.
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