Oxidative decarboxylation of pyruvate by 1-deoxy-D-xyulose 5-phosphate synthase, a central metabolic enzyme in bacteria

Article abstract of DOI:10.1074/jbc.RA118.001980

The underexploited antibacterial target 1-deoxy-D-xyluose 5-phosphate (DXP) synthase catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate and D-glyc-eraldehyde 3-phosphate (D-GAP). DXP is an essential intermediate in the biosynthesis of ThDP, pyridoxal phosphate, and isoprenoids in many pathogenic bacteria. DXP synthase catalyzes a distinct mechanism in ThDP decarboxylative enzymology in which the first enzyme-bound pre-decarboxylation intermediate, C2α-lactyl-ThDP (LThDP), is stabilized by DXP synthase in the absence of D-GAP, and D-GAP then induces efficient LThDP decarboxylation. Despite the observed LThDP accumulation and lack of evidence for C2α-carbanion formation in the absence of D-GAP, CO₂ is released at appreciable levels under these conditions. Here, seeking to resolve these conflicting observations, we show that DXP synthase catalyzes the oxidative decarboxylation of pyruvate under conditions in which LThDP accumulates. O₂-dependent LThDP decarboxylation led to one-electron transfer from the C2α-carbanion/enamine to O₂, with intermediate ThDP-enamine radical formation, followed by peracetic acid formation en route to acetate. Thus, LThDP formation and decarboxylation and DXP formation were studied under anaerobic conditions. Our results support a model in which O₂-dependent LThDP decarboxylation and peracetic acid formation occur in the absence of D-GAP, decreasing the levels of pyruvate and O₂ in solution. The relative pyruvate and O₂ concentrations then dictate the extent of LThDP accumulation, and its buildup can be observed when [pyruvate] > [O₂]. The finding that O₂ acts as a structurally distinct trigger of LThDP decarboxylation supports the hypothesis that a mechanism involving small molecule– dependent LThDP decarboxylation equips DXP synthase for diverse, yet uncharacterized cellular functions.

Full text of DOI:10.1074/jbc.RA118.001980

JBC Papers in Press. Published on May 21, 2018 as Manuscript RA118.001980
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA118.001980
Oxidative decarboxylation of pyruvate by 1-deoxy-D-xyulose 5-phosphate synthase, a central metabolic
enzyme in bacteria
Alicia A. DeColli1, Natasha S. Nemeria2, Ananya Majumdar3, Gary J. Gerfen4, Frank Jordan2,
Caren L. Freel Meyers1*
1Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of
Medicine, Baltimore, MD, 21205, United States
2Department of Chemistry, Rutgers University, Newark, NJ, 07102, United States
3 Department of Chemistry, Johns Hopkins University, Baltimore, MD, 21218, United States
4 Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY, 10461,
United States
Running title: O2-dependent LThDP decarboxylation on DXP synthase
*To whom correspondence should be addressed: C. Freel Meyers: Department of Pharmacology and
Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205, United
States; cmeyers@jhmi.edu; Tel. (410) 502-4807; FAX. (410) 955-3023.
Keywords: DXP synthase, decarboxylase, oxygen binding, oxidation-reduction, redox, thiamine, enzyme
mechanism, pyridoxal phosphate, oxygenase, oxidative decarboxylation
Abstract
The underexploited antibacterial target 1-
model
in
which
O2-dependent
LThDP
decarboxylation and peracetic acid formation occur
in the absence of D-GAP, decreasing the levels of
pyruvate and O2 in solution. The relative pyruvate
and O2 concentrations then dictate the extent of
LThDP accumulation, and its buildup can be
observed when [pyruvate] > [O2]. The finding that
O2 acts as a structurally distinct trigger of LThDP
decarboxylation supports the hypothesis that a
mechanism involving small molecule-dependent
LThDP decarboxylation equips DXP synthase for
diverse, yet uncharacterized cellular functions.
deoxy-D-xyluose 5-phosphate (DXP) synthase,
catalyzes the thiamin diphosphate (ThDP)-
dependent formation of DXP from pyruvate and D-
glyceraldehyde 3-phosphate (D-GAP). DXP is an
essential intermediate in the biosynthesis of ThDP,
pyridoxal phosphate, and isoprenoids in many
pathogenic bacteria. DXP synthase catalyzes a
distinct mechanism in ThDP decarboxylative
enzymology in which the first enzyme-bound pre-
decarboxylation intermediate, C2α-lactylThDP
(LThDP), is stabilized by DXP synthase in the
absence of D-GAP, and D-GAP then induces
efficient LThDP decarboxylation. Despite the
observed LThDP accumulation and lack of
evidence for C2α-carbanion formation in the
absence of D-GAP, CO2 is released at appreciable
levels under these conditions. Here, seeking to
resolve these conflicting observations, we show
that DXP synthase catalyzes the oxidative
decarboxylation of pyruvate under conditions in
which LThDP accumulates. O2-dependent LThDP
decarboxylation led to one-electron transfer from
the C2α-carbanion/enamine to O2, with
intermediate ThDP-enamine radical formation,
followed by peracetic acid formation en route to
Introduction
The increasing spread of antimicrobial
resistance demands the development of novel
antimicrobial agents. 1-deoxy-D-xyluose 5-
phosphate (DXP) synthase is an underexploited
antimicrobial target that catalyzes the formation of
DXP from pyruvate and D-glyceraldehyde 3-
phosphate (D-GAP, Figure 1) in many human
pathogens (e.g. Mycobacterium and Plasmodium
falciparum). DXP is an essential branch point
metabolite that serves as a precursor in the
biosynthesis of thiamin diphosphate (ThDP),
pyridoxal
phosphate,
and
indispensable
isoprenoids in bacterial pathogens (Figure 1) (1-4).
Given the widespread essentiality of DXP in
pathogens and its absence in humans, selective
acetate.
Thus,
LThDP
formation
and
decarboxylation and DXP formation were studied
under anaerobic conditions. Our results support a
1

O2-dependent LThDP decarboxylation on DXP synthase
inhibition of DXP synthase could offer a new
antimicrobial strategy (5-8).
DXP synthase catalyzes oxidative decarboxylation
of pyruvate by a mechanism involving O2-
dependent LThDP decarboxylation and subsequent
oxidation of the C2-carbanion to produce
peracetic acid as an intermediate in acetate
formation. Removal of O2 does not significantly
impact the rates of LThDP formation, D-GAP-
dependent LThDP decarboxylation, or DXP
formation suggesting O2 does not influence the
DXP-forming activity of DXP synthase. However,
the stability of LThDP depends upon the presence
or absence of O2, indicating that mechanistic
studies of LThDP formation and decarboxylation in
the absence of D-GAP should be conducted under
anaerobic conditions. This study provides key
insights into our understanding of DXP synthase
mechanism and raises interesting questions about
the function of the unique requirement for ternary
complex formation on DXP synthase.
DXP synthase possesses pyruvate
decarboxylase and carboligase activity (Figure 1),
reminiscent of other ThDP-dependent enzymes (9).
However, its distinct structure (10-12) and unique
random sequential, preferred order mechanism (13-
17) distinguish DXP synthase from other enzymes
in this class, including its closest mammalian
homologs, transketolase and the E1 component of
pyruvate dehydrogenase (10). In contrast to other
ThDP-dependent enzymes that utilize classical
ping-pong kinetics, DXP synthase stabilizes
LThDP and requires ternary complex formation
upon binding of D-GAP to trigger efficient
decarboxylation of LThDP by an unknown
mechanism (13, 15). These mechanistic insights
have guided selective inhibitor design (5-8).
The requirement for ternary complex
formation during catalysis was first proposed based
14
upon CO2 trapping studies demonstrating a low
Results
rate of CO2 release in the absence of D-GAP (13).
Circular dichroism (CD) and NMR studies
subsequently revealed the accumulation of LThDP
on DXP synthase in the absence of D-GAP, and
provided evidence that D-GAP plays a role to
induce efficient LThDP decarboxylation (15).
These findings are consistent with a mechanism
involving ternary complex formation on DXP
synthase; however, these results do not explain the
earlier observation that DXP synthase catalyzes
multiple turnovers and CO2 release in the presence
of excess pyruvate. DXP synthase-dependent
acetolactate formation was demonstrated (18),
providing a possible explanation for the observed
CO2 release in the absence of D-GAP. However, the
acetolactate synthase activity of DXP synthase was
later shown to be exceedingly inefficient (15).
LThDP stabilization on DXP synthase, coupled
with inefficient acetolactate formation and a lack of
evidence for C2α-carbanion generation in the
absence of D-GAP (by detection of acetaldehyde
formation from hydroxyethylThDP (HEThDP) or
2,6-dichlorophenolindophenol (DCPIP) reduction,
Figure 2) (15) are collectively inconsistent with the
extent of CO2 release observed by Eubanks and
Poulter (13). Given the unique requirement for a
trigger of LThDP decarboxylation on DXP
synthase, resolving this discrepancy is important.
Here, we report studies that reconcile these
Product formation on DXP synthase in the
absence of D-GAP
To obtain a comprehensive profile of
products formed by the action of DXP synthase in
the absence of D-GAP, the DXP synthase reaction
using uniformly 13C3-pyruvate was monitored over
time by NMR. 1D 13C (Figure 3) and 2D HCC-
COSY experiments (Figure S1) experiments
revealed enzyme-dependent depletion of pyruvate
(resonances at δ 204.97 ppm (C1, dd), 170.13 ppm
(C2, dd), 26.47 (C3, dd)) with concomitant
formation of acetate (resonances at δ 181.41 ppm
(C1, d) and 23.31 ppm (C2, d)) and bicarbonate (δ
160.36 ppm (s)) as the major products, indicating
that CO2 release occurs under conditions in which
accumulation of LThDP is evident by CD analysis
(Figures 3A-3C). Orthogonal biochemical assays
were conducted to measure CO2 release and
pyruvate consumption. A 14CO2 trapping assay was
conducted as previously described (13), and
showed DXP synthase and pyruvate-dependent
14CO2 release over time, consistent with
bicarbonate formation detected by NMR (Figure
3D). Concomitant with CO2 release, DXP
synthase-dependent pyruvate consumption was
observed by the lactate dehydrogenase (LDH)
assay (Figure 3E). Taken together, these results
indicate that DXP synthase catalyzes the oxidative
decarboxylation of pyruvate in the absence of D-
conflicting observations. Our results indicate that
2

O2-dependent LThDP decarboxylation on DXP synthase
GAP. These findings are consistent with the
Electron paramagnetic resonance (EPR)
studies of DXP synthase in the presence of
evidence for CO2 release reported by Eubanks and
Poulter (13), yet puzzling in light of the CD
evidence for LThDP accumulation and D-GAP-
dependent LThDP decarboxylation on DXP
synthase under these conditions (15). Thus,
experiments were conducted toward understanding
oxidative decarboxylation and determining the
trigger for CO2 release on DXP synthase under
these conditions.
13
2
unlabeled, C3 and H3 labeled pyruvate revealed
the formation of a radical species at concentrations
of approximately 1% of DXP synthase active
centers (Figure 6, solid lines). The spectra
generated with 2H3-pyruvate (Figure 6A) displayed
an observable narrowing with respect to the
unlabeled sample (Figure 6B), as would be
expected if the electron spin of the radical species
experienced a decreased (albeit unresolved)
hyperfine coupling to the deuterons derived from
2H3-pyruvate relative to the protons of the
unlabeled pyruvate. Moreover, spectra generated
with 13C3-pyruvate produced a spectrum (Figure
6C) with observable broadening with respect to the
unlabeled sample, which is expected if the radical
experienced the additional (unresolved) hyperfine
coupling to the 13C nuclei (nuclear spin ½) which is
absent in the 12C (nuclear spin = 0) sample.
Oxidative decarboxylation of pyruvate proceeds
via the ThDP-enamine radical and peracetic acid
formation
Several routes were considered for DXP
synthase-catalyzed acetate formation (Figure 4).
These were based on previous reports of ThDP-
dependent enzymes that catalyze the oxidative
decarboxylation of pyruvate (19-21) and those that
form radical ThDP intermediates (22-27).
Following decarboxylation of LThDP, one-
electron transfer from the C2α-carbanion/enamine
1 to molecular oxygen results in the formation of a
C2-(α-hydroxy)-ethylideneThDP radical cation
(ThDP-enamine radical) 2 and superoxide. In path
A (Figure 4), recombination of 2 with superoxide
would generate ThDP-bound peroxide intermediate
3. Elimination of cofactor from 3 would yield
peracetic acid which is susceptible to rapid
chemical decomposition to acetate in HEPES, pH 8
(Figure S2) (20, 28). Elimination of H2O2 from 3
(path B, Figure 4) would afford acetylThDP 4,
which could undergo hydrolysis to release acetate
and cofactor. Finally, in path C (Figure 4), a second
electron transfer from 2 to superoxide could occur
resulting in the production of H2O2 and 4 en route
to acetate. Notably, only LThDP has been detected
on DXP synthase by NMR following an acid
quench, with no detectable formation of
acetylThDP 4 (15, 16).
The impact of the labeled pyruvate on the
EPR spectrum of the radical species clearly
implicates the contribution of carbon and hydrogen
derived from pyruvate to the structure of the radical
species. To determine if the EPR spectra are
consistent with the ThDP-enamine radical species
2 (Figure 4), simulations were generated (Figure 6,
dashed lines) using parameters (Table S1)
previously determined for similarly structured
radical species generated by other ThDP-dependent
enzymes (22-26). All parameters were held
constant in the three simulations, with the
exception of the appropriate addition or scaling of
hyperfine interactions for the labeled nuclei
previously determined (22-26). The fits provide
strong evidence that the radical intermediate
generated by DXP synthase is consistent with 1-
electron oxidation of the enzyme-bound C2α-
carbanion intermediate 1 to form 2 en route to
acetate.
Each proposed pathway to acetate requires
To distinguish between paths A and B/C
O2 consumption and formation of a radical ThDP
intermediate (2, Figure 4). To confirm that O2 is
utilized as a substrate by DXP synthase, the
concentration of O2 was monitored over time in the
presence of varying concentrations of DXP
synthase and pyruvate. Indeed, enzyme and
pyruvate-dependent O2 consumption was detected
providing further support for DXP synthase-
catalyzed oxidative decarboxylation of pyruvate
(Figure 5, Table 1).
(Figure 4), we utilized two previously described
biochemical assays that specifically monitor
peracetic acid (path A) or H2O2 (paths B or C)
formation (20, 29). DXP synthase-catalyzed
peracetic acid formation was probed using a
coupled assay with thionitrobenzoate (TNB, max
=
412 nm) which is readily oxidized by peracetic acid
at a significantly higher rate (~3000-fold higher)
compared to its oxidation by H2O2 (20), thus
providing a selective detection method to
3

O2-dependent LThDP decarboxylation on DXP synthase
differentiate between peracetic acid and H2O2
To determine if O2 acts as a trigger of CO2
production. Control experiments were conducted to
confirm selective detection of peracetic acid under
reaction conditions used here (Figure S3).
Observation of DXP synthase-dependent oxidation
of TNB (Table 1) suggests that peracetic acid is
formed in the oxidative decarboxylation of
pyruvate by DXP synthase. Further, the rate of
DXP synthase-dependent peracetic acid formation
is comparable to the rate of O2 consumption under
the same conditions (Table 1), suggesting that
peracetic acid formation coincides with O2
consumption, similar to acetolactate synthase and
pyruvate decarboxylase (20). In an attempt to
ascertain whether acetate formation occurs via
either path B or C, we employed a discontinuous
assay for selective detection of H2O2 (29).
Unfortunately, control experiments conducted
during this study indicate that H2O2 is unstable
under these reaction conditions and, therefore,
cannot be accurately detected by this method
(Figure S4). Although DXP synthase-catalyzed
H2O2 formation cannot be definitively ruled out,
the comparable rates of TNB and O2 consumption
(Table 1) and lack of acetylThDP detection on DXP
synthase to date support path A as the major route
to acetate.
release, we conducted the CO2 trapping and
pyruvate consumption assays under anaerobic
conditions, in the presence or absence of added O2
(established by purging reaction mixtures in an
environment of 90 % N2/10 % H2 mix). Under
anaerobic conditions in the absence of D-GAP, CO2
release and pyruvate consumption are significantly
decreased (Figures 8A and 8B), suggesting O2 is
required for efficient release of CO2 in the absence
of D-GAP. The observed low levels of CO2 release
and pyruvate consumption under anaerobic
conditions could result from incomplete removal of
O2 or slow acetolactate formation. Addition of O2
to the anaerobic sample results in significant CO2
release (Figure 8A), providing direct evidence for
O2-induced decarboxylation. Importantly, under
anaerobic conditions, addition of a deoxygenated
D-GAP solution results in significant CO2 release
which is consistent with our previous
characterization of D-GAP as a trigger of efficient
LThDP decarboxylation on DXP synthase (Figure
8A) (15).
Behavior of LThDP on DXP synthase under
aerobic and anaerobic conditions
If the model depicted in Figure 7 is correct,
The newly revealed understanding that
the stabilization of LThDP on DXP synthase
should be observed by CD under anaerobic
conditions, and the LThDP species should be
unstable in the presence of a trigger of CO2 release
(O2 or D-GAP). As expected, addition of pyruvate
to DXP synthase under anaerobic conditions leads
to the emergence of a positive CD signal at 313 nm
(Figure 9A) which has previously been
DXP
synthase
catalyzes
the
oxidative
decarboxylation of pyruvate suggests a model
(Figure 7) to explain the simultaneous observations
of LThDP accumulation and CO2 release in the
absence of D-GAP. In the presence of O2, LThDP
decarboxylation occurs by an unknown
mechanism, to form the C2α-carbanion which is
then oxidized to form peracetic acid (Figure 7).
This reaction cycle consumes pyruvate and O2 and
depletes a proportion of LThDP-bound DXP
synthase; thus, the relative concentrations of O2 and
pyruvate dictate the extent to which LThDP
accumulation and/or oxidative decarboxylation is
observed. Over time, DXP synthase deoxygenates
characterized as LThDP on DXP synthase (15).
pyruvate
The apparent Kd
under anaerobic conditions
is 45 ± 2 µM (Table 2). As illustrated in Figure 9B,
in the absence of added triggers, following
saturation of DXP synthase with pyruvate, the
LThDP signal is stable for > 4 h. Immediate
depletion of the signal at 313 nm is observed upon
addition of a deoxygenated solution of D-GAP
(1000 M, Figure 9C) or upon aeration of the
sample (via opening the lid and mixing, Figure 9D),
which further supports both D-GAP and O2 as
triggers of LThDP decarboxylation and is
consistent with the model shown in Figure 7.
Importantly, this suggests that under aerobic
conditions, the reactive nature of the LThDP
intermediate may lead to unreliable measurements
the solution to
a
point where LThDP
decarboxylation no longer occurs efficiently,
permitting stabilization of LThDP over a longer
time scale. To provide support for this model, we
conducted a series of experiments to determine the
effect of O2 on the activity of DXP synthase.
Characterization of triggers of CO2 release on
DXP synthase
4

O2-dependent LThDP decarboxylation on DXP synthase
of Kdpyruvate, depending upon the extent of O2
introduction through mixing. Varied aeration will
change the amount of pyruvate required for DXP
synthase to effectively deoxygenate the solution
and permit observation of significant LThDP
accumulation. In contrast, pyruvate is not turned
over when measurements are conducted in an
0.05 s-1) (15), showing O2 does not significantly
impact the kinetics of LThDP formation on DXP
synthase (Figure 11, Table 3). It is interesting to
note that under anaerobic conditions, following
addition of pyruvate to the pre-formed DXP
synthase-ThDP complex, there is a short lag phase
(Figure 11, inset, region 1) followed by a decrease
in the CD signal at 313 nm (Figure 11, inset, region
2) that immediately precedes the signal increase
corresponding to LThDP formation (Figure 11,
inset, region 3). This behavior of the CD signal was
not observed under aerobic conditions (10), and
could signify binding and/or conformational
change(s) occurring on DXP synthase prior to
LThDP formation in the absence of O2.
anaerobic environment, permitting a more reliable
pyruvate
measurement of the apparent Kd
.
Steady state kinetic characterization of DXP
formation under anaerobic conditions
DXP formation is the only known
physiological function of DXP synthase, and our
results indicate the possibility that O2 may compete
with D-GAP in the LThDP decarboxylation and/or
carboligation steps leading to DXP formation.
Thus, we investigated the steady-kinetics of DXP
formation in the absence of O2 to determine the
effect of O2 on the component steady state kinetic
parameters, Km and kcat, of wild type E. coli DXP
synthase. The results indicate that there is no
significant difference in the kinetic parameters
measured in the presence or absence of O2 (Figure
10, Table 2).
A significant decrease in the CD313 signal
upon the addition of a trigger to pre-formed LThDP
is interpreted to coincide with LThDP
decarboxylation (15). It follows that the CD signal
corresponding to pre-formed LThDP should be
minimally affected by the addition of deoxygenated
buffer lacking O2 or D-GAP triggers required for
efficient LThDP decarboxylation. Consistent with
this, a marginal change in the CD313 signal is
observed upon rapid mixing of deoxygenated
buffer in one syringe with pre-formed LThDP in a
second syringe under anaerobic conditions (Figure
12A), in contrast to the significant decrease at 313
nm observed in the presence of D-GAP or O2
(Figures 12B and 12C). The subtle change in CD
signal detected in the absence of added trigger
(Figure 12A) is likely due to incomplete removal of
O2 during the deoxygenation process, leading to
decarboxylation of a small fraction of LThDP.
While an accurate background rate of LThDP
decarboxylation could not be determined in this
case, apparent LThDP re-synthesis (k1r) is
suggested by the data (app k1r = 1.04 ± 0.11 s-1). In
contrast, the rates of LThDP decarboxylation (k2)
upon addition of O2-saturated buffer or
deoxygenated buffer containing D-GAP to
preformed LThDP (prepared under anaerobic
conditions) were 62.0 ± 8.9 s-1 and 50.3 ± 7.8 s-1,
respectively (Figures 12A-12C, Table 3),
comparable to the rate of LThDP decarboxylation
in the presence of D-GAP under aerobic conditions
previously reported (k2 = 42 ± 1 s-1) (15). These
results suggest that LThDP decarboxylation occurs
efficiently in the presence of either D-GAP or O2.
Notably, previously observed lag phases in the
CD313 signal occurring immediately before and
Pre-steady state characterization of LThDP
formation and decarboxylation under anaerobic
conditions
Steady state CD analysis of LThDP
accumulation on DXP synthase in the presence and
absence of O2 provides support for a model in
which the stability of the LThDP signal depends on
the level of O2 in solution. This suggests that
experiments measuring accumulation or depletion
of LThDP may be confounded by O2-dependent
LThDP decarboxylation and pyruvate consumption
under aerobic conditions. Therefore, the pre-steady
state rates of LThDP formation and
decarboxylation (in the presence of deoxygenated
buffer, D-GAP, or O2-saturated buffer) were
determined to obtain more accurate measurements
of these kinetic parameters. To determine the rate
of LThDP formation on DXP synthase in the
absence of O2, DXP synthase in one syringe was
rapidly mixed with pyruvate in the other syringe
under anaerobic conditions. The results indicate a
rate of 1.64 ± 0.21 s-1 for LThDP formation (k1) on
DXP synthase under anaerobic conditions that is
comparable to the previously reported rate of
LThDP formation under aerobic conditions (1.39 ±
5

O2-dependent LThDP decarboxylation on DXP synthase
after the sharp decrease in this signal corresponding
to LThDP decarboxylation (16) are observed upon
addition of D-GAP or O2-saturated buffer to
preformed LThDP generated under anaerobic
conditions (Figures 12B and 12C, insets). These lag
phases are proposed to signify conformational
changes occurring prior to LThDP decarboxylation
and immediately after LThDP decarboxylation
prior to LThDP re-synthesis (16), a hypothesis that
is supported by recent HDX-MS studies (12).
enzyme. The oxidative decarboxylation of pyruvate
is a key element of DXP synthase mechanism that
explains the accumulation of LThDP and lack of
accumulation of the C2α-carbanion, despite
significant CO2 release (13, 15). Our inability to
detect post-decarboxylation intermediates on DXP
synthase in the absence of D-GAP, is likely a
consequence of electron transfer from the C2α-
carbanion to O2 preventing efficient reaction of this
intermediate with other potential acceptor
substrates. The simultaneous observations of
LThDP accumulation and CO2 release can be
explained by the model in Figure 7. Under
conditions where O2 is in excess to pyruvate,
occurrence of LThDP decarboxylation at a lower
rate than C2α-carbanion consumption, would
permit the observation of the LThDP intermediate
concomitant with CO2 release and peracetic
acid/acetate formation. The presence of O2 ensures
this cycle of LThDP decarboxylation and peracetic
acid formation, consuming both pyruvate and O2
(Figure 7). As a result, the relative concentrations
of O2 and pyruvate dictate the extent of buildup and
the length of time for which LThDP is stable.
Therefore, accumulation of LThDP over a long
time period requires conditions where pyruvate is
in excess to O2, which is the case for previously
reported CD and NMR experiments (15). Under
Discussion
Although important aspects of the DXP
synthase mechanism are uncovered, gaps in our
understanding of catalysis on this unique ThDP-
dependent enzyme remain. The present study
addresses a discrepancy in mechanistic studies of
DXP synthase: the observation of slow CO2 release
in the absence of a known trigger LThDP
decarboxylation, D-GAP (13), despite the
accumulation of LThDP on the enzyme and lack of
evidence for the post-decarboxylation intermediate
(15). Our results reveal two roles of O2: 1) as a
distinct trigger of LThDP decarboxylation, and 2)
as an acceptor substrate in the oxidation of the C2α-
carbanion to form peracetic acid en route to acetate.
Other ThDP-dependent enzymes are known to
catalyze similar reactions (19-21, 30). However,
due to the ability of DXP synthase to stabilize
LThDP, and the lack of any indication that C2α-
carbanion formation takes place in the absence of
D-GAP, this oxygen-consuming reaction was not
previously characterized. The results show that
steady state and pre-steady state kinetic parameters
are unchanged under anaerobic conditions
suggesting O2 does not affect catalytic steps prior
these
conditions,
enzyme-catalyzed
O2
consumption deoxygenates the solution, resulting
in a net accumulation of LThDP in the absence of
O2-dependent decarboxylation, reconciling the
discrepancy in our understanding of DXP synthase
mechanism.
Although the requirement of a small-
molecule (e.g. D-GAP or O2) to trigger efficient
LThDP decarboxylation on DXP synthase is
established, the mechanism by which this occurs is
not fully elucidated. Several mechanistic studies of
DXP synthase have revealed compelling evidence
for D-GAP-dependent conformational changes (12,
16, 17). For example, a recent hydrogen-deuterium
exchange mass spectrometry (HDX-MS) study
showed that a donor substrate analog and D-GAP
induce closed and open conformations of DXP
synthase, respectively (12). It is conceivable that D-
GAP binding induces a conformational change that
destabilizes the LThDP intermediate, lowering the
barrier for chemical decarboxylation of LThDP
(12, 15, 16). Considering the proposal that 2-
oxoacid decarboxylation is reversible on ThDP-
to and including DXP formation. The apparent
pyruvate
Kd
measured under anaerobic conditions is a
more accurate estimation as anaerobic conditions
prevent confounding reactions that occur in the
presence of O2 (i.e., depletion of pyruvate and
LThDP). Although the oxygenase activity of DXP
synthase does not appear to affect DXP-forming
activity of wild type enzyme, this side reaction and
discovery
of
O2-dependent
LThDP
decarboxylation have important implications for
the study of DXP synthase mechanism.
In retrospect, the observation of LThDP
buildup on DXP synthase was fortuitous given the
efficiency of decarboxylation in the presence of O2,
and points to the unique mechanistic aspects of this
6

O2-dependent LThDP decarboxylation on DXP synthase
function of the unique mechanism of DXP synthase
dependent enzymes (31-33), it is also possible that
an equilibrium between LThDP and the C2α-
carbanion/CO2 is established on DXP synthase in a
conformation that limits release of CO2 from the
enzyme. Subsequent binding of D-GAP could then
drive the equilibrium triggering LThDP depletion
and turnover. These mechanistic models cannot be
distinguished by the tools used in this study.
Although O2 is structurally and chemically distinct
from D-GAP, it appears to trigger depletion of
LThDP with a comparable rate and imparts similar
behavior to the CD313 signal of the LThDP species
(Figure 12), suggesting a similar mechanism of
LThDP decarboxylation is at play in the presence
of these two triggers. Further studies are required
to determine if O2 can induce a conformational
change on DXP synthase, toward understanding the
mechanism by which O2 and D-GAP induce
LThDP decarboxylation and release of CO2, and to
determine if other natural triggers exist.
in the broader context of microbial metabolism.
Experimental Procedures
General methods
Unless otherwise noted, all reagents were
obtained from commercial sources. Aerobic
spectrophotometric analyses were performed on a
Beckman DU800 UV/Visible spectrophotometer
(Brea, CA, USA). Anaerobic spectrophotometric
analyses were performed on a Varian Cary 50
UV/Visible spectrophotometer (Palo Alto, CA,
USA). Anaerobic conditions were established
either in a Baker-Ruskinn Invivo (Sanford, ME,
USA) or a Coy Laboratory Products vinyl
anaerobic chamber (Grass Lake, MI, USA). E. coli
wild-type DXP synthase and E. coli MEP synthase
(IspC) were overexpressed and purified as reported
previously with minor modifications for the
purification of anaerobic DXP synthase. (14, 34)
To obtain anaerobic DXP synthase, the protein was
overexpressed and purified as previously described
(34) however the second dialysis was carried out in
an anaerobic chamber in 50 mM 4-(2-
Moving forward, it is imperative to
consider the O2-dependent E-LThDP depletion in
mechanistic studies of DXP synthase. First, we
have shown that addition of O2 to LThDP by
sample mixing causes depletion of the CD313 signal,
indicating that Kd determinations for donor
substrates should be performed under anaerobic
conditions to obtain the most accurate
measurement. Second, it is imperative that studies
aimed at identifying other inducers of LThDP
decarboxylation or investigating the roles of active
site residues in catalysis, are conducted under
anaerobic conditions to avoid interfering side
reactions or variable contributions of oxygenase
activity on variants, both of which could confound
results. Other challenges to investigating DXP
synthase, such as structural studies, may also be
resolved by working under anaerobic conditions.
In this study, we have uncovered a novel
activity of DXP synthase, adding to the repertoire
of chemistry that this unique enzyme can catalyze.
Notably, we illustrate that O2 is a structurally
distinct trigger of LThDP decarboxylation which
has important mechanistic and technical
implications for the study of DXP synthase
mechanism. These findings add a major piece to the
mechanistic puzzle of DXP synthase and have
enabled resolution of inconsistencies in the
literature. Taken together, these results lay a
foundation to gain a deeper understanding of the
hydroxyethyl)-1-piperazineethanesulfonic
acid
(HEPES), pH 8, 5% glycerol, 100 mM NaCl, 10
mM MgCl2, and 1 mM ThDP at 0 ºC for 4 h. The
protein was stored in liquid N2 until use when it was
transferred directly to an anaerobic chamber.
Radioactivity measurements where made with a
Beckman LS6500 scintillation counter (Brea, CA,
USA). X-band (9 GHz) EPR measurements were
made using a Varian E-112 spectrometer equipped
with a TE102 cavity and interfaced to a PC using
custom written software. Steady-state CD spectra
were recorded on an Aviv 420 CD spectrometer
(Lakewood, NJ, USA). Pre-steady state CD spectra
were recorded on an Applied Photophysics Pi*-180
stopped-flow CD spectrometer (Leatherhead,
Surrey, UK) with a 10-mm path length cuvette.
NMR spectra were obtained on a Bruker Avance III
500 MHz NMR spectrometer (Billerica, MA,
USA). Oxygen consumption was measured with
the Oxytherm + respiration oxygen monitoring
system from Hansatech Instruments Ltd (Norfolk,
UK).
NMR detection of product formation by DXP
synthase in the absence of D-GAP
7

O2-dependent LThDP decarboxylation on DXP synthase
To monitor reaction progress by NMR,
DXP synthase (30 µM) was mixed with 13C3-
pyruvate (500 µM) in 50 mM HEPES-D18, pH 8,
100 mM NaCl, 1 mM ThDP, 2 mM MgCl2, 1 %
glycerol, and 10% D2O to initiate product
counting time per sample). DPMs were determined
from the measured CPMs using the counting
efficiency determined from a standard curve of [1-
14C]-pyruvate in the acidic medium or [14C]-
bicarbonate in the basic medium. All reactions
were carried out in triplicate. Because this quench
and trapping protocol varied slightly from that of
Eubanks and Poulter (13), a trapping control was
conducted to confirm that >95 % of the 14CO2 that
is released from the enzyme is collected in our trap
(Figure S5).
13
formation. 1D C spectra (500 MHz, 256 scans,
0.7 s/scan) were recorded over time at 25 °C. A
control lacking enzyme was prepared in the same
buffer and incubated at 25 °C for 4 h after which a
spectrum was recorded. Spectra of 13C2-acetate and
13C-bicarbonate standards were obtained in the
NMR buffer and with the same acquisition
parameters.
Lactate dehydrogenase detection of pyruvate
consumption under aerobic conditions
14CO2 trapping under aerobic conditions in the
absence of D-GAP
For lactate dehydrogenase (LDH)
detection of pyruvate consumption, reactions
contained 30 µM DXP synthase and 500 µM
pyruvate in reaction buffer (described above).
These solutions were preincubated for 5 min at 0
°C in the absence of DXP synthase. Pyruvate
consumption was initiated upon addition of DXP
synthase or storage buffer for the no enzyme
control. At 0.5, 5, and 60 min, a 60 µL aliquot of
the reaction solution was boiled at 100 °C for 5 min
to quench the reaction. Precipitated protein was
pelleted by centrifugation. The pyruvate remaining
in the quenched solution was then determined using
LDH. Reactions (400 µL) containing 40 µL of the
supernatant from the quenched reaction solution,
200 µM NADH, 32.5 mM KH2PO4, pH 7.4, and
0.75 U/mL yeast LDH were initiated upon addition
of the pyruvate-containing solution and the
absorbance at 340 nm (ε = 6220 M-1 cm-1) was
monitored at 37 °C for 15 min. The amount of
pyruvate in each sample was calculated using the
change in the initial and final absorbance.
Corrections were made for non-enzymatic
oxidation of NADH over the course of the LDH
reaction. DXP synthase reactions were conducted
in triplicate.
To measure 14CO2 release from DXP
synthase, the same general protocol from Eubanks
and Poulter was conducted with some minor
modifications (13). Briefly, reactions (250 µL)
contained 30 µM DXP synthase and [1-14C]-
pyruvate diluted with cold pyruvate (0.18
µCi/µmol total pyruvate, 500 µM pyruvate total) in
reaction buffer (50 mM HEPES, pH 8, 100 mM
NaCl, 1 mM ThDP, 2 mM MgCl2, and 1 %
glycerol). Solutions were pre-incubated in the
absence of DXP synthase at 0 °C for 5 min in the
outer well of a trapping vial (glass vial, 4.5 cm
height and 2.5 cm diameter, with 24/40 ground
glass neck and a center well trap, 2 cm height and
1.5 cm diameter). The center well trap contained
500 µL of 1 M NaOH, and a 24/40 septum stopper
was used to seal the trapping vial. Reactions were
then initiated upon addition of DXP synthase to the
outer well via syringe and incubated at 0 °C. At 0.5,
5, and 60 min, 250 L of 12.5 % TCA in 1M HCl
was added to the outer well of the trapping vial via
syringe to quench the reaction and drive hydrated
14CO2 out of solution (note each timepoint was a
separate reaction). A negative control in which
enzyme storage buffer (50 mM Tris, pH 8, 10 %
glycerol, 100 mM NaCl, 10 mM MgCl2, 1 mM
ThDP) was added in place of enzyme was also
prepared and “quenched” at 60 min. The quenched
solutions were incubated at 25 °C for 2 h to trap the
14CO2 released by DXP synthase. The entire
contents of the acid quench solution and the NaOH
trap solution were added to 10 mL of Hionic-Fluor
scintillation fluid (Perkin Elmer), mixed by
vortexing, and immediately counted (1-min
Detection of oxygen consumption
To determine if DXP synthase consumes
O2 in an enzyme- and substrate-dependent manner,
DXP synthase concentration was varied (0- 30 µM)
in the presence of 500 µM pyruvate, and pyruvate
concentration was varied (0-500 µM) in the
presence of 5 µM DXP synthase in reaction buffer
(described above). Solutions were prepared in the
absence of pyruvate, added to the electrode
8

O2-dependent LThDP decarboxylation on DXP synthase
chamber (calibrated fresh each day at 25 °C) and
equilibrated at 25 °C for at least 4 min until the O2
signal stabilized. O2 consumption was initiated
upon addition of pyruvate (25 µL regardless of the
final [pyruvate]) by syringe, and the concentration
of O2 was monitored over time at 25 °C. O2
consumption was monitored under each condition
in triplicate.
14CO2 trapping under anaerobic conditions in the
presence and absence of triggers
For the detection of 14CO2 under anaerobic
conditions the general method used for the aerobic
14CO2 trapping assay was performed with some
modifications. Before addition of the [1-14C]-
pyruvate, all solutions and the trapping vials
(prepared with 500 µL of 1 M NaOH in the center
well) were deoxygenated in an anaerobic chamber
for 15 min at 30 ºC and then sealed with septa
before removing from the chamber. [1-14C]-
Pyruvate was added to the reaction solution which
was distributed to the outside wells of the trapping
vials via syringe. One trapping vial was purged
with 100 % O2 for 10 min at 0 ºC before addition of
the reaction solution. All trapping vials were then
incubated at 0 ºC for 5 min. 14CO2 release was
initiated upon addition of DXP synthase, and the
mixtures were incubated at 0 ºC for 1 h. The
reactions were then quenched, trapped, and counted
as described for aerobic conditions.
Electron prarmagnetic reasonance detection of
ThDP-enamine radical species
DXP synthase (32 mg/mL, 508 µM) in 0.4
ml of 50 mM HEPES (pH 8.0) containing 0.10 M
NaCl, 1.0 mM ThDP, 10 mM MgCl2 and 10 %
glycerol was mixed with unlabeled pyruvate (10
2
13
mM), H3-pyruvate (10 mM), or C3-pyruvate (10
mM) at room temperature. Mixtures were
immediately transferred into EPR tubes and flash-
frozen in liquid nitrogen after a 30-40 s incubation
at room temperature. During EPR measurements,
the sample temperature was held at 77 K using an
immersion finger dewar. Spectrometer parameters
used to acquire the spectra are as follows:
modulation amplitude: 5 G; microwave power, 10
µW; microwave frequency, 9.09 GHz; scan time, 2
min; time constant, 0.5 s; number of scans, 9. The
field was calibrated using a standard sample of
manganese doped in MgO (35). The concentration
of active sites occupied by the ThDP radical species
was calculated by comparing the double integral of
the radical EPR spectrum with both Cu(II) and
nitroxide standard sample spectra (23).
LDH detection of pyruvate consumption under
anaerobic conditions
The same general method for LDH
detection of pyruvate under aerobic conditions was
repeated for a single 1-h timepoint under anaerobic
conditions. Samples were prepared in the absence
of DXP synthase, and both the reaction solutions
and DXP synthase were purged in septa-capped
Eppendorf tubes in the anaerobic chamber for 15
min at 30 ºC. All solutions were capped before
removing from the chamber, and incubated at 0 ºC
for 5 min. Pyruvate consumption was initiated upon
addition of DXP synthase by syringe. After 1 h at 0
ºC, the samples were transferred to 100 ºC to
quench the reactions. Precipitated DXP synthase
was removed by centrifugation, and the pyruvate
remaining in the supernatant was determined using
the LDH assay as described above. Reactions were
carried out in triplicate.
Colorimetric detection of DXP synthase-
dependent peracetic acid formation
To determine if DXP synthase catalyzes
the formation of peracetic acid as an intermediate
in acetate formation, we utilized the previously
described TNB assay (20). Samples were prepared
in the reaction buffer described above with 75 µM
TNB (synthesized and characterized according to
the protocol in the SI) varying DXP synthase (0- 30
µM) in the presence of 500 µM pyruvate or varying
pyruvate (0-500 µM) in the presence of 5 µM DXP
synthase. After a 5-min preincubation at 25 ºC,
peracetic acid formation was initiated upon
addition of DXP synthase and the oxidation of TNB
was monitored spectrophotometrically (λmax = 412
nm, ε = 13,600 M-1 cm-1) at 25 ºC over time. All
reactions were carried out in triplicate.
Steady state CD analysis of DXP synthase under
anaerobic conditions
DXP synthase (30 µM) in 50 mM HEPES,
pH 8, 100 mM NaCl, 1 mM MgCl2, 1 mM BME,
0.2 mM ThDP (3 mL total) was deoxygenated in an
anaerobic chamber for 30 min at 25 ºC. The sample
was transferred to a septa-capped cuvette (Starna
Cells, 1-Q-10-ST-S) which was closed before
9

O2-dependent LThDP decarboxylation on DXP synthase
removal from the chamber. A DXP synthase only
scan was obtained from 450-280 nm at 4 ºC with a
1 nm step and 1.6 s averaging time. Pyruvate (7-
401 µM) was titrated into the sample via syringe,
and the CD spectrum after each addition of
pyruvate was recorded as described above. The
titration was carried out in triplicate. D-GAP (1
mM) or O2 (via aeration of the sample by pipette)
were added after the final pyruvate addition, and a
spectrum obtained by the scan method described
above. The normalized CD signal at 313 nm,
indicative of LThDP on DXP synthase (15), was
plotted against the concentration of pyruvate to
to room air. For both the aerobic and anaerobic
characterizations, the depletion of NADPH (λmax
=
340 nm, ε = 6220 M-1 cm-1) was monitored over
time at 25 ºC following initiation of DXP
formation. The rates of NADPH depletion were
used to determine the initial rates of DXP
formation. Plots of initial rate of DXP formation
against [substrate] were fit to the Michaelis-Menten
equation in GraphPad Prism Version 6. Reactions
were performed in duplicate.
Pre-steady state CD characterization of LThDP
formation under anaerobic conditions
pyr
For all pre-steady state experiments, the
obtain an apparent Kd and the plot was fit to a
CD signal of LThDP at 313 nm was monitored at 6
ºC over time. All samples were prepared with
anaerobic stocks (prepared from solids with
deoxygenated water in an anaerobic chamber) in a
portable glove box (Belart) under constant flow of
100 % N2. Trace oxygen inside the glove box was
removed with Anaero-Packs (Mitsubushi) and the
O2 level the chamber was monitored with Anaero-
one-site specific binding curve in GraphPad Prism
Version 6. All Kd determinations were performed
in triplicate.
Steady state kinetic characterization of DXP
formation under aerobic and anaerobic
conditions
DXP formation was monitored using the
indicator
tablets
(Mitsubushi).
Anaerobic
previously described DXP synthase-IspC coupled
assay with some modifications (14). Pyruvate
concentration was varied (6.25-1000 µM) while
holding D-GAP constant at 500 µM, or D-GAP
concentration was varied (3.125- 1000 µM) while
holding pyruvate constant at 1 mM with 100 nM
DXP synthase, 2 µM IspC, and 200 µM NADPH in
100 mM HEPES, pH 8, 5 mM NaCl, 2.5 mM
TCEP, 2 mM MgCl2, and 1 mM ThDP. When D-
GAP concentration was varied, samples were
prepared without pyruvate and initiated with
pyruvate. When pyruvate concentration was varied,
samples were prepared without pyruvate or D-GAP,
and enzymatic reactions were initiated with both
substrates. For the anaerobic characterization,
solutions were prepared from anaerobic stocks in
an anaerobic chamber at 26 ºC (either prepared
from solids in the chamber with deoxygenated
water or solutions made anaerobic by incubation in
the chamber for 15 min before sample preparation).
Reaction solutions in septa-topped cuvettes (Starna
cells, 9B-Q-10-GL14-S) and initiation solutions in
septa-topped vials were sealed before removal from
the chamber. Reactions where initiated upon
addition of substrate via syringe as described
above, and the solutions were mixed by tilting the
cuvettes. Samples for the aerobic characterization
were treated similarly but with constant exposure
conditions were established and maintained in the
stopped flow CD using the anaerobic accessory and
anaerobic operation protocol provided by Applied
Photophysics. (36) To determine the rate of LThDP
formation, DXP synthase (60 µM) in buffer A (50
mM HEPES, pH 8, 1 mM ThDP, 100 mM NaCl, 2
mM MgCl2) was rapidly mixed with an equal
volume of pyruvate (2 mM) in buffer A. The CD
signal at 313 was monitored for 10 s at 6 ºC and the
data from 7 repetitive shots were averaged. The
data from 0.01 - 10 s were fit to a double
exponential association model (Equation 1) using
SigmaPlot version 10:
y(t) = y0 + a* (1-exp(-k1t)) + b*(1-exp(-k1’t))
Equation 1
where y0 is the CD313 at t = 0, k1 and k1’ represent
the rate constants of the faster and slower phases of
LThDP formation respectively, and a and b
describe the maximum CD313 for the faster and
slower phases, respectively. Error represents the
error of the curve fit.
Pre-steady state CD characterization of
decarboxylation rates in the presence and absence
of triggers under anaerobic conditions
10

O2-dependent LThDP decarboxylation on DXP synthase
The effect of O2 on the rates of LThDP
decarboxylation and resynthesis in the presence
and absence of D-GAP were determined by
conducting the experiments under anaerobic
conditions. For the rate of LThDP decarboxylation
in the absence of triggers, LThDP was preformed
in one syringe (60 µM DXP synthase and 1 mM
pyruvate in buffer A described above) and mixed
with an equal volume of buffer A in a second
syringe under anaerobic conditions. The CD signal
at 313 nm was monitored at 6 ºC for 5 s and the data
where y0 is the CD313 at t = 0, a describes the
minimum CD313, and k2 represents the rate constant
for LThDP decarboxylation. For the determination
of LThDP decarboxylation rates, data points
between 0.023 and 0.3 s were used in the (+) O2
analysis and data points between 0.023 and 0.165 s
were used in the (+) D-GAP analysis. Rates of
LThDP resynthesis were determined using either
the double exponential association model
(Equation 1) for the (+) D-GAP experiment or a
single association model (Equation 3) for the (+) O2
experiment using SigmaPlot version 10:
from
7
repetitive shots were averaged.
Determinations of the rates of LThDP
decarboxylation in the presence of D-GAP and O2
were conducted similarly except the second syringe
contained either 1 mM D-GAP or oxygenated
buffer A (prepared by bubbling 100% O2 through 1
mL of buffer A for 1 h at 25 ºC), respectively. The
data for LThDP decarboxylation were fit to a single
exponential decay model (Equation 2) using
SigmaPlot version 10:
y(t) = y0 + a* (1-exp(-k1rt))
Equation 3
where y0 is the CD313 at t = 0, a describes the
maximum CD313, and k1r represents the rate
constant for LThDP resynthesis. Data points
between 0.358 and 5 s where used for the analysis
of LThDP resynthesis in the presence of O2 and
data points between 0.2 and 5 s where used in the
analysis of LThDP resynthesis in the presence of D-
GAP. Error represents the error of the curve fit
y(t) = y0 + a * exp(-k2t)
Equation 2
Acknowledgments
The authors thank the Liz Calzada (Claypool Lab, JHMI), Risa Burr (Espenshade Lab, JHMI),
and Erica Sinner (Townsend Lab, JHU) for kindly allowing access to instruments and advice on the study
of oxygen consumption and work under anaerobic conditions. We are grateful to Melanie Johnston for her
helpful discussion throughout the study, and her generous aid in establishing anaerobic techniques. We
thank the JHU Biomolecular NMR Center for 600 MHz data collection. This work was supported by the
National Institutes of Health grant to C.L.F.M R01 GM084998.
Conflict of Interest
The authors declare that they have no conflicts of interest with the contents of this article. The
content is solely the responsibility of the authors and does not necessarily represent the official views of
the National Institutes of Health.
Contributions
C.L.F.M. and A.A.D. conceived and designed the study and wrote the paper. A.A.D conducted
and analyzed data for most of the experiments. N.S.N and A.A.D. performed the experiments and
analyzed the data in Figures 11-12 and Table 3. A.M. performed the experiment and analyzed the data in
Figure S1 and provided technical assistance and method development for the data in Figures 3B-C.
N.S.N. and G.J.G. designed the experiment for Figure 6. G.J.G. conducted the experiment and analyzed
the data for Figure 6. N.S.N., A.M., G.J.G, and F.J. contributed to the preparation of the manuscript. All
authors reviewed the results and approved the final version of the manuscript.
11

O2-dependent LThDP decarboxylation on DXP synthase
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O2-dependent LThDP decarboxylation on DXP synthase
Footnotes
This work was supported by the National Institutes of Health grant to C.L.F.M R01 GM084998. The
authors declare that they have no conflicts of interest with the contents of this article. The content is
solely the responsibility of the authors and does not necessarily represent the official views of the
National Institutes of Health.
Abbreviations
DXP, 1-deoxy-D-xylulose 5-phosphate; ThDP, thiamin diphosphate; D-GAP, D-glyceraldehyde 3-
phosphate; LThDP, C2α-lactylThDP; CD, circular dichroism; DCPIP, 2,6-dichlorophenolindophenol;
HEThDP, hydroxyethylThDP; ThDP-enamine radical, C2-(α-hydroxy)-ethylideneThDP radical cation;
DXPS, DXP synthase; EPR, electron paramagnetic resonance, TNB, thionitrobenzoate; HDX-MS,
hydrogen-deuterium exchange mass spectrometry, HEPES, 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid; LDH, lactate dehydrogenase.
15

O2-dependent LThDP decarboxylation on DXP synthase
Table 1. Rates of peracetic acid formation in the presence of varying concentrations of DXP synthase and
500 µM pyruvate at 25 ºC are comparable to rates of O2 consumption under the same conditions
supporting Path A (Figure 4) as the major route for oxidative decarboxylation of pyruvate. Error
represents standard error, n = 3
Table 2. Summary of steady state kinetic and binding parameters determined for DXP synthase under
anaerobic conditions at 25 °C. Error represents standard deviation, n = 2.
16

O2-dependent LThDP decarboxylation on DXP synthase
Table 3. Summary of pre-steady state kinetic parameters of DXP synthase in the presence and absence of
D-GAP under aerobic and anaerobic conditions.
17

O2-dependent LThDP decarboxylation on DXP synthase
Figure 1. DXP synthase catalyzes the formation of the essential branch point metabolite, DXP, from
pyruvate and D-GAP.
Figure 2. Reactions of the C2α-carbanion intermediate are exceedingly slow in the absence of D-GAP at
37 ºC (15) indicating the C2α-carbanion intermediate does not build up on DXP synthase.
18

O2-dependent LThDP decarboxylation on DXP synthase
-
Figure 3. A) DXP synthase-mediated pyruvate consumption to form acetate and HCO3 . B) and C) 13C
NMR time course of the reaction of 30 µM DXP synthase with 3 mM 13C3-pyruvate at 25 ºC in the
absence of D-GAP (a. 3 min, b. 1 h, c. 4 h). At 4 h, 13C2-acetate and 13C-bicarbonate (3 mM) were added
to the reaction mixture (d). Pyruvate is not consumed in the absence of DXP synthase (e). Spectra were
acquired of 13C2-acetate and 13C-bicarbonate (f and g). D) 14CO2 release is detected in a reaction
containing 30 µM DXP synthase and [1-14C]-pyruvate (0.18 µCi/µmol pyruvate, 500 µM total pyruvate)
at 0 ºC. E) Pyruvate (500 µM) consumption in the presence of 30 µM (7.2 nmol) DXP synthase at 0 ºC is
detected by the LDH assay. Error bars represent standard error, n = 3
19

O2-dependent LThDP decarboxylation on DXP synthase
Figure 4. Possible routes for acetate formation via DXP synthase-catalyzed oxidative decarboxylation of
pyruvate.
Figure 5. DXP synthase-dependent O2 consumption is observed at 25 ºC in the presence of varying DXP
synthase (DXPS) concentrations and pyruvate (500 µM). Representative data shown (n = 3).
20

O2-dependent LThDP decarboxylation on DXP synthase
Figure 6. X-band (9 GHz) CW-EPR spectra of the radical species generated by DXP synthase (508 µM)
in the presence of 10 mM 2H3-pyruvate (A), unlabeled pyruvate (B), or 13C3-pyruvate (C). Solid lines
represent experimental data and dashed lines are simulations generated using parameters given in Table
S1 which are consistent with the radical structure 2, Figure 4. Simulation details are provided in the
supplementary information.
Figure 7. Proposed model for oxidative decarboxylation of pyruvate illustrating the roles of O2 as 1) a
trigger for CO2 release, and 2) an acceptor substrate in peracetic acid formation. This catalytic cycle
results in depletion of O2, pyruvate, and LThDP, and the amount of LThDP accumulation and/or peracetic
acid formation is expected to depend on the relative levels of O2 and pyruvate in a given sample.
21

O2-dependent LThDP decarboxylation on DXP synthase
Figure 8. A) Detection of 14CO2 release following incubation of DXP synthase (30 µM) and [1-14C]-
pyruvate (0.18 µCi/µmol pyruvate, 500 µM pyruvate total) for 60 min at 0 ºC under anaerobic conditions
(90 % N2/ 10 % H2), in the presence or absence of O2 (septa-capped trapping vial purged with 100 % O2
for 10 min prior to addition of the reaction solution) or D-GAP (1 mM). B) LDH detection of pyruvate
remaining in solution after a 60 min incubation of pyruvate (500 µM, 32.5 nmol) in the presence or
absence of DXP synthase (30 µM, 1.95 nmol) at 0 ºC under anaerobic conditions. Error bars represent
standard error, n = 3
22

O2-dependent LThDP decarboxylation on DXP synthase
Figure 9. A) Titration of pyruvate onto DXP synthase (30 µM) at 4 ºC under anaerobic conditions reveals
an apparent Kdpyruvate of 45 ± 2 µM (Error represents standard error, n = 3). A representative titration is
shown). B) The CD signal at 313 nm (characteristic of LThDP) is stable on DXP synthase under
anaerobic conditions for > 4 h at 4 ºC. Addition of D-GAP (C) or aeration (D) of a sample of preformed
LThDP results in depletion of the LThDP CD signal at 313 nm.
23

O2-dependent LThDP decarboxylation on DXP synthase
Figure 10. Representative Michaelis-Menten curves for DXP synthase determined with the IspC-coupled
assay at 25 ºC varying D-GAP under anaerobic (A) or aerobic conditions (B) and varying pyruvate under
anaerobic (C) or aerobic conditions (D).
Figure 11. Pre-steady state formation of LThDP on DXP synthase (30 µM) in the presence of 1 mM
pyruvate under anaerobic conditions at 6 ºC. A lag (region 1) and a decrease (region 2) in the CD signal at
313 nm are observed upon addition of pyruvate to the DXP synthase-ThDP complex prior to LThDP
formation (region 3). Data points are an average of the CD signal over time from 7 repetitive shots. Error
is calculated from the fit of the averaged data to a double exponential rise to a maximum (see Equation 1
in Materials and Methods)
24

O2-dependent LThDP decarboxylation on DXP synthase
Figure 12. Pre-steady state decarboxylation of LThDP at 6 ºC upon addition of anaerobic buffer (A), an
anaerobic solution of D-GAP (B), or O2 saturated buffer (C) to preformed LThDP under anaerobic
conditions. Insets show up to the first 300 ms of the full time course to illustrate the lags in the CD signal
at 313 nm before and after LThDP decarboxylation. Data points are an average of the CD signal over time
from 7 repetitive shots. Error is calculated from the fit of the averaged data to the appropriate equation
(see Materials and Methods)
25

Oxidative decarboxylation of pyruvate by 1-deoxy-D-xyulose 5-phosphate synthase, a
central metabolic enzyme in bacteria
Alicia A. DeColli, Natasha S. Nemeria, Ananya Majumdar, Gary J. Gerfen, Frank Jordan
and Caren L. Freel Meyers
J. Biol. Chem. published online May 21, 2018
Access the most updated version of this article at doi: 10.1074/jbc.RA118.001980
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