DXP Synthase-Catalyzed CN Bond Formation: Nitroso Substrate Specificity Studies Guide Selective Inhibitor Design

Article (PDF Available)inChemBioChem 14(11) · July 2013with29 Reads
DOI: 10.1002/cbic.201300187 · Source: PubMed
1-Deoxy-D-xylulose 5-phosphate (DXP) synthase catalyzes the first step in the nonmammalian isoprenoid biosynthetic pathway to form DXP from pyruvate and D-glyceraldehyde 3-phosphate (D-GAP) in a thiamin diphosphate-dependent manner. Its unique structure and mechanism distinguish DXP synthase from its homologues and suggest that it should be pursued as an anti-infective drug target. However, few reports describe any development of selective inhibitors of this enzyme. Here, we reveal that DXP synthase catalyzes CN bond formation and exploit aromatic nitroso substrates as active site probes. Substrate specificity studies reveal a high affinity of DXP synthase for aromatic nitroso substrates compared to the related ThDP-dependent enzyme pyruvate dehydrogenase (PDH). Results from inhibition and mutagenesis studies indicate that nitroso substrates bind to E. coli DXP synthase in a manner distinct from that of D-GAP. Our results suggest that the incorporation of aryl acceptor substrate mimics into unnatural bisubstrate analogues will impart selectivity to DXP synthase inhibitors. As a proof of concept, we show selective inhibition of DXP synthase by benzylacetylphosphonate (BnAP).



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Available from: Jürgen Bosch, Sep 16, 2015
DOI: 10.1002/cbic.2013001 87
DXP Synthase-Catalyzed CN Bond Formation: Nitroso
Substrate Specificity Studies Guide Selective Inhibitor
Francine Morris,
Ryan Vierling,
Lauren Boucher,
Jrgen Bosch,
Caren L. Freel Meyers*
The isoprenoids are a vast and structurally diverse class of nat-
ural products derived from two simple bioprecursors, isopen-
tenyl diphosphate (IDP) and dimethylallyl diphosphate
(DMADP). Essential in all living organism s, isoprenoids are bio-
synthesized through two distinct pathways. The mevalonate
pathway for IDP and DMADP biosynthesis is found in mam-
mals and fungi. In contrast, most human pathogens, including
many bacterial pathogens
and the malaria parasite, Plasmodi-
um falciparum,
are known to use the methylerythritol phos-
phate (MEP) pathway (Scheme 1) for the generation of IDP and
Its essentiality and prevalence in human pathogens,
and absence in mammals, renders the MEP pathway a target
for the development of new anti-infective agents, which are
desperately needed to combat the emergence and re-emer-
gence of drug resistance.
Seven biosynthetic steps make up the MEP pathway, begin-
ning with the formation of 1-deoxy-d-xylulose 5-phosphate
(DXP) from pyruvate (donor substrate) and d-glyceraldehyde 3-
phosphate (d-GAP, acceptor substrate). This first transformation
is catalyzed by DXP synthase in a thiamin diphosphate (ThDP)-
dependent manner,
and is believed to play a regulatory role
in isoprenoid biosynthesis.
In addition, DXP synthase is a
branch point in pathogen metabolism.
Its product, DXP, is
required for IDP/DMADP biosynthesis and is also a precursor in
pyridoxal biosynthesis and, notably, ThDP biosynthesis, whic h
is required for the formation of DXP itself.
The importance of DXP synthase in pathogen metabolism
highlights this enzyme as a particularly interesting new drug
target. Additionally, DXP synthase is mechanistically distinct
from other ThDP-dependent enzymes. The enzyme combines
decarboxylase and carboligase chemistry in a ThDP-dependent
condensation of pyruvate and d-GAP (Scheme 1). A report by
Eubanks et al.
provided compelling evidence for a unique
catalytic mechanism in which binding of both acceptor and
donor substrates is required to induce decarboxylation of pyru-
vate to form a kinetica lly competent ternary complex. In subse-
quent studies,
we have provided further support for the
formation of a ternary complex during DXP synthase catalysis
and d-GAP-promoted decarboxylation of the C2 a-lactylthia-
1-Deoxy-d-xylulose 5-phosphate (DXP) synthase catalyzes the
first step in the nonmammalian isoprenoid biosynthetic path-
way to form DXP from pyruvate and d-glyceraldehyde 3-phos-
phate (d-GAP) in a thiamin diphosphate-dependent manner.
Its unique structure and mechanism distinguish DXP synthase
from its homologues and suggest that it should be pursued as
an anti-infective drug target. However, few reports describe
any development of selective inhibitors of this enzyme. Here,
we reveal that DXP synthase catalyzes CN bond formation
and exploit aromatic nitroso substrates as active site probes.
Substrate specificity studies reveal a high affinity of DXP syn-
thase for aromatic nitroso substrates compared to the related
ThDP-dependent enzyme pyruvate dehydrogenase (PDH). Re-
sults from inhibition and mutagenesis studies indicate that
nitroso substrates bind to E. coli DXP synthase in a manner dis-
tinct from that of d-GAP. Our results suggest that the incorpo-
ration of aryl acceptor substrate mimics into unnatural bisub-
strate analogues will impart selectivity to DXP synthase inhibi-
tors. As a proof of concept, we show selective inhibition of
DXP synthase by benzylacetylphosphonate (BnAP).
Scheme 1. Biosynthesis of isoprenoids IDP and DMADP through the methyl-
erythritol phosphate (MEP) pathway.
[a] F. Morris, R. Vierling, Prof. C. L. Freel Meyers
Department of Pharmacology and Molecular Sciences
The Johns Hopkins University School of Medicine
725 North Wolfe St, Baltimore, MD 21205 (USA)
E-mail: cmeyers@jhmi.edu
[b] L. Boucher, Prof. J. Bosch
Department of Biochemistry and M olecular Biology
The Johns Hopkins Malaria Research Institute
Bloomberg School of Public Health
615 North Wolfe Street, Baltimore, MD 21205 (USA)
Supporting information for this article is available on the WWW under
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 1309 1315 1309
min diphosphate (LThDP) intermediate, the pre-decarboxyla-
tion intermediate formed by reaction of pyruvate and ThDP. In
contrast, all other ThDP-dependent enzymes are believed to
follow classical ping-pong kinetics in which the activation of
pyruvate and the release of carbon dioxide precede binding of
the acceptor substrate.
In addition, structural analysis indi-
cates that DXP synthase has a unique domain arrangement in
which the active site is between domains of the same mono-
mer within the homodimer.
This is in contrast to its homo-
logues, in which the active site is at the dimer interface.
Taken together, these observations highlight unique aspects of
DXP synthase catalysis and structure that distinguish it from its
mammalian homologues, and suggest that it should be possi-
ble to selectively target this enzyme so as to develop new
anti-infective agents. However, reports describing the develop-
ment of selective DXP synthase inhibitors are scarce,
due to a perception that selective inhibition of DXP synthase
over mammalian ThDP-dependen t enzymes will be difficult,
We have pursued substrate specificity studies of DXP syn-
thase to try to reveal important substrate binding determi-
nants that could guide selective inhibitor design. Previously,
we have shown that aliphatic aldehydes are accepted as alter-
native substrates to give the corresponding a-hydroxy ke-
this suggests that DXP synthase displays some flexi-
bility toward nonphosphorylated acceptor substrates. A subse-
quent study revealed the selective inhibitory activity of a series
of alkylacetylphosphonates designed to act as unnatural bisub-
strate analogues targeting a conformation of DXP synthase
that uniquely accommodates both a donor and acceptor sub-
strate in the formation of a ternary complex.
The largest
alkylacetylphosphonate, butylacetylphosphonate, exhibited
greater selectivity of inhibition than ethyl- and methylacetyl-
phosphonates, thus indicating that selective targeting of DXP
synthase is possible.
In this study, we explore the capacity of DXP synthase to
bind sterically demanding scaffolds by evaluating its usage of
aromatic acceptor substrates. We demonstrate the capacity of
DXP synthase to catalyze the formation of CN bonds to gen-
erate aromatic hydroxamic acids or amides from nitroso sub-
strates. The intrinsically higher reactivity of nitroso substrate
analogues compared to their aldehyde counterparts has per-
mitted a substrate specificity study revea ling aromatic sub-
strates with high affinity for the enzyme. Further, our results
suggest aromatic substrates might adopt a different binding
mode from d-GAP in a relatively large active site compared to
that of pyruvate dehydrogenase (PDH) or transketolase (TK).
These results have prompted the design and synthesis of
a DXP synthase inhibitor bearing an aromatic component to
impart selectivity.
Aromatic aldehydes as DXP synthase substrates
Some ThDP-dependent enzymes are known to catalyze CC
bond formation by using aromatic substrates with various
turnover efficiencies;
however, there are no reports de-
scribing the use of aromatic substrates by DXP synthase. We
tested several aromatic aldehydes as acceptor substrates, of
which 2-hydroxy-4,6-dinitrobenzaldehyde appeared to be
amongst the best, this was therefore fully characterized as
a substrate for DXP synthase (Figure S1). It has a K
of (512
20) mm, approximately 18 times higher than that of the natural
substrate, d-GAP, and its k
is low (k
= (0.350.05) min
The aromatic aldehyde study suggested that there might be
flexibility in the active site of DXP synthase toward aromatic
acceptor substrates (data not shown). However, a significant
number of aromatic aldehydes are not turned over by DXP
synthase; this suggests that the low intrinsic reactivity of aro-
matic aldehydes is a limiting factor in substrate specificity
studies to probe the enzyme active site.
DXP synthase-catalyzed CN bond formation
The ni troso group is a functional isostere of the aldehyde
group and is known to possess higher reactivity toward nucle-
ophiles. In fact, the ThDP-utilizing enzymes TK, pyruvate decar-
boxylase (PDC), benzaldehyde lyase (BAL) and PDH have been
shown to use aromatic nitroso analogues as acceptor sub-
strates in the formation of hydroxamic acids.
We hy-
pothesized that a substrate specificity study of DXP synthase
with the intrinsically more reactive aromatic nitroso compound
class would better inform us about key binding elements of ar-
omatic substrates. In addition, we postulated that such a study
could reveal a new application of DXP synthase as a biocatalyst
for the generation of the medicinally important hydroxamic
acid class.
Thus, a series of aromatic nitroso analogues was tested as
substrates for DXP synthase. Notably, DXP synthase turns over
a range of structurally diverse nitroso substrates (1, 39,
Scheme 2); most aldehyde counterparts of these nitroso ana-
logues are not substrates for the enzyme; this is consistent
Scheme 2. Nitroso substrate usage by DXP synthase.
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 1309 1315 1310
with the idea that the nitroso isostere is more reactive. A rep-
resentative HPLC stack plot that illustrates DXP synthase-cata-
lyzed conversion of the simplest aromatic nitroso analogue, ni-
trosobenzene (1), to the corresponding hydroxamic acid (2)is
shown in Figure 1 (see also Figure S2). A single C-nitroso ana-
logue, 10, did not act as substrate for the enzyme; neither
were N-nitroso compounds substrates. The electron-rich p-di-
methylamino nitroso analogue 4 is a substrate for DXP syn-
thase (Figure S4), but not for yeast TK.
Interestingly, the cor-
responding amides, presumably produced through a mecha-
nism involving the unstable hydroxamic acid as an intermedi-
were detected as the major products of several elec-
tron-rich substrates (48, Scheme 2, Figures S4–S8)). This result
has been reported in a study that examined the turnover of 4
by PDC.
However, the observation that the amide is also
isolated from naphthol substrates was unexpected. In order to
rule out the possibility that bovine serum albumin (BSA) added
to enzymatic reaction mixtures catalyzes the formation of
amide products, control reactions were performed on 4 and 5
in the absence of BSA. In both cases, only the corresponding
amides were detected.
Kinetic parameters were measured spectrophotometrically
for the alternative substrates shown in Table 1. The specificity
constant k
for nitrosobenzene is of the same order of
magnitude as that of the natural acceptor substrate, d-GAP. Re-
duced specificity constants were measured for the larger naph-
thol-containing substrates 5 8 (Table 1), an observation that is
consistent with the idea that sterically demanding naphthol
substrates could exhibit a reduction in efficiency of turnover as
a consequence of reduced affinity for the enzyme. However,
detailed kinetic analysis of nitroso substrate turnover suggests
this is not the case. Small nitrosobenzene analogues display
higher reactivity (high k
) but lower affinity (higher K
) rela-
tive to d-GAP (1 and 3, Table 1). Contrary to our expectations,
several sterically demanding alternative substrates exhibit high
affinities for DXP synthase, with nitrosonaphthols 58 showing
comparable affinity to the natural substrate. In these cases,
a reduced k
accounts for the lower turnover efficiency, in line
with previous reports on the sensitivity of nitroso turnover to
substituent effects.
The remarkably high affinities measured
for sterically demanding substrates on DXP synthase is in stark
contrast to previously reported trends in nitroso turnover by
ThDP-dependent enzymes;
here increasing steric bulk of the
substrate correlated with decreased affinity.
Aromatic nitroso substrates exhibit low affinity for the
smaller PDH active site
As a basis for selective inhibitor design, we determined wheth-
er DXP synthase displays a higher affinity for sterically de-
manding substrates than PDH does. Thus, nitroso analogues 1,
4 and 6 were evaluated as substrates for porcine PDH (Figures
S10 and S11). Our results indicate these aromatic substrates
exhibit significantly lower affinities for PDH than for DXP syn-
thase (Table 2), in contrast to the trend observed for DXP syn-
thase. Nitrosobenzene displays a 2.7-fold increase in K
PDH compared to DXP synthase, whereas the largest of the
nitroso substrates tested, nitrosonaphthol 6, displays an ap-
Figure 1. HPLC analysis of the DXP-synthase-dependent conversion of nitro-
sobenzene to hydroxamic acid 2.
Table 1. Substrate specificity of nitroso substrates.
Substrate k
d-GAP 1027284 36460
1 17519 13318 13220
3 3679916 369
4 0.90.1 5413 1.70.5
5 1.10.2 4110 2.70.8
6 2.00.2 24682
7 1.180.04 184 6.61.5
8 1.30.2 637 2.10.4
9 1.40.2 38718 0.360.05
[a] Reaction conditions: 100 mm HEPES, pH 8.0, 2 mm MgCl
,5mm NaCl,
1mm ThDP, 1 mgmL
BSA, 10–20 mm pyruvate, 10 % DMSO (v/v), 378C.
[b] Performed in triplicate. Values shown are the averageSEM.
Table 2. Determination of K
for nitroso substrates against E. coli DXP
synthase (DXPS) compared to the porcine PDH E1 subunit.
Substrate PDH K
1 35030 2.7
4 40860 7.5
6 45016 19.3
[a] Reaction conditions: 100 mm HEPES, pH 8.0, 2 mm MgCl
,5mm NaCl,
1mm ThDP, 1 mgmL
BSA, 10–20 mm pyruvate, 10 % DMSO (v/v), 378C.
Performed in triplicate; values shown are the average SEM.
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 1309 1315 1311
proximately 19-fold increase in K
for PDH compared to DXP
synthase. We hypothesized that the DXP synthase active site
might be relatively larger to accommodate ternary complex
formation during catalysis. Indeed, a comparison of active site
volumes (calculated from crystal structure coordinates that
were aligned in Coot
and then analyzed by using Pocket-
) suggests the DXP synthase active site is significantly
larger than those of the ThDP-dependent enzymes PDH or
transketolase (Figures 2 and S12). The hydrophobic nature of
the alternative nitroso substrates tested could potentially drive
the selectivity of turnover by DXP synthase. However, when
the active site pockets of DXP synthase, PDH, and TK were ana-
lyzed by using fpocket,
the computed hydrophobicity score
(based on the hydrophobicity scale published by Monera
et al.
) indicated that the PDH pocket is more hydrophobic
than that of DXP synthase, whereas TK has the least hydropho-
bic pocket (Table S1). Taken together, these results suggest
that the incorporation of sterically demanding fragments into
inhibitor scaffolds could drive selective inhibition and is facili-
tated mostly by the larger cavity of DXP synthase.
Inhibition of DXP formation by nitroso alternative
The low K
values measured for aromatic nitroso substrates
suggest that these analogues bind with reasonable affinity in
the enzyme active site. Thus, we hypothesized that alternative
substrates with aromatic scaffolds could also act as inhibitors
of the natural reaction. Compounds 1 and 39 were evaluated
as inhibitors of DXP synthase by using an HPLC-based assay
previously reported.
Interestingly, all the nitroso compounds
exhibited weak inhibitory activity with IC
values ranging from
208 mm to > 2mm and with no apparent trend with measured
values (Table 3). As one of the hi gher-affinity substrates, the
readily available nitrosonaphthol, 5, was selected for further
evaluation in an effort to understand the mechanism of inhibi-
tion. This inhibitor was found to exhibit a competitive inhibi-
tion pattern with respect to d-GAP (apparent K
80) mm, Figure S14). The more than tenfold difference between
the K
((4110) mm) and K
suggests that nitrosonaphthols
could adopt a binding mode for turnover that is distinct from
the binding mode for inhibition. Alternatively, K
might reflect
the affinity of the Michaelis–Menten complex between enzyme
and nitrosonaphthol, whereas the K
for this substrate could
be indicative of a higher-affinity ternary complex further along
the reaction coordinate in this two-substrate system.
Nitrosonaphthols and d-GAP adopt distinct binding modes
during turnover
R478 and R420 are known to be essential for the binding of d -
GAP, presumably by anchoring the phosphate group (unpub-
lished results). Two DXP synthase variants (R478A and R420A)
were evaluated as catalysts for CN bond formation with nitro-
sonaphthols 57. Although both of these mutations adversely
affect the binding of d-GAP, they have no apparent effect on
the affinities of nitroso substrates in CN bond formation, as
indicated by the comparable K
values measured for both var-
iant and wild-type enzymes with nitroso substrates (Table 4,
Figure S15). This is consistent with the notion that nitroso-
naphthols adopt a binding mode for turnover that is distinct
from that of d-GAP.
Selective inhibition of DXP synthase by benzyl acetyl-
phosphonate (BnAP)
Our results suggest that the comparatively large active site of
DXP synthase can accommodate sterically demanding scaf-
folds, but in a manner that does not interfere with DXP forma-
tion. On this basis, we hypothesized that aromatic components
could be incorporated into unnatural bisubstrate analogues to
Figure 2. Overlay of the DXP synthase, transketolase (TK), and PDH E1 sub-
unit active site pockets. The DXP synthase active site is predicted to be
larger than the other two.
Table 3. Inhibition of DXP formation by nitroso substrates.
Substrate IC
Substrate IC
1 20820 6 52260
3 29111 7 35490
4 844170 8 >2000
5 1065190 9 >2000
=(42280) mm)
[a] Reaction conditions: 100 mm HEPES, pH 8.0, 2 mm MgCl
,5mm NaCl,
1mm ThDP, 1 mg mL
BSA, 10 % DMSO (v/v), 30 mmd-GAP, 80 mm pyru-
vate, 37 8C. [b] Performed in triplicate; values shown are the average
Table 4. WT, R478A, and R420A catalyze comparable turnovers of nitroso-
Alternative K
substrate WT DXP synthase R478A
5 4110 234325
6 246203161
7 184131142
[a] Reaction conditions: 100 mm HEPES, pH 8.0, 2 mm MgCl
,5mm NaCl,
1mm ThDP, 1 mgmL
BSA, 10–20 mm pyruvate, 10 % DMSO (v/v), 378C.
[b] Performed in triplicate; values shown are the average SEM.
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 1309 1315 1312
impart selectivity of inhibition against DXP synthase. To dem-
onstrate this concept, we prepared benzyl acetylphosphonate
(BnAP) as a potential selective inhibitor of DXP synthase. BnAP
incorporates the acetyl phosphonate moiety as a pyruvate
mimic and a benzyl group to mimic the alternative acceptor
substrate, nitrosobenzene (Figure 3A). As expected, BnAP is a
competitive inhibitor with respect to pyruvate with reasonable
potency against DXP synthase (K
=(10.41.3) mm), and exhib-
its ~85-fold higher inhibitory activity against DXP synthase
than PDH (Figure 3). Additionally, BnAP exhibits an uncompeti-
tive inhibition pattern with respect to d-GAP (K
= (708) mm,
Figure 3D). The requirement for d-GAP binding is consistent
with the idea that aromatic scaffolds adopt a binding mode
that is distinct from d-GAP.
DXP synthase is an attractive drug target for the development
of new anti-infective agents, and selective inhibitors of this
enzyme are sought. Our study highlights CN bond formation
as a new reaction catalyzed by DXP synt hase and shows ni-
troso substrates to be useful tools for probing the active site
of this potential drug target. Our study shows that DXP syn-
thase-catalyzed CN bond formation can lead to the genera-
tion of hydroxamic acids and amides, with electron-rich nitroso
substrates giving predominantly amide products. Although the
mechanism for this transformation has not been elucidated, it
is thought to occur through a hydroxamic acid intermediate.
Notably, we have demonstrated that nitroso substrate ana-
logues with a naphthol scaffold exhibit an exceptional affinity
for DXP synthase that is comparable to that of the natural
acceptor substrate, d-GAP. Further, sterically demanding sub-
strates are selectively turned over by DXP synthase
and show a considerably lower affinity for the ThDP-
dependent enzyme PDH. Consistent with this finding,
calculations indicate that the volume of the DXP syn-
thase active site is significantly greater than that of
PDH or transketolase and can thus accommodate
sterically demanding alternative substrates. The alter-
native acceptor substrates tested in this study are
surprisingly weak inhibitors of DXP formation, with
nitrosonaphthol (5) acting as a weak competitive in-
hibitor against d-GAP. The more than tenfold discrep-
ancy between K
and K
for this compound suggests
that multiple binding modes could be possible for 5,
or they could reflect a lower-affinity complex en
route to a higher-affinity ternary complex (described
by K
). Evidence that nitrosonaphthols
adopt a different binding mode from d-GAP during
turnover was obtained through substitution of R478
and R420, active site residues that are essential for d-
GAP binding. The variant s R478A and R420A display
efficient turnover and comparable affinity for nitroso-
naphthols to wild-type DXP synthase. Further studies
are needed to define the critical residues for CN
bond formation, and especially those that are impor-
tant for aromatic substrate binding.
Taken together, the data suggest that incorporat-
ing an aromatic group into an unnatural bisubstrate
analogue scaffold should give the analogue the abili-
ty to selectively inhibit DXP synthase over other
ThDP-dependent enzymes. Indeed, benzylacetyl-
phosphonate selectively inhibits DXP synthase with
a K
of (10.41.3) mm and K
~85. Although BnAP is
comparable in inhibitory activity to butylacetylphosphonate,
an increase in K
is observed; this suggests sterically
demanding aromatic acetylphosphonates as a promising new
class of selective DXP synthase inhibitors.
Experimental Section
General: Unless otherwise noted, all reagents were obtained from
commercial sources. HPLC analyses were performed on a Beckman
Gold Nouveau System with a Grace Alltima 3 mm C18 analytical
Rocket column (53 mm 7 mm). Spectrophotometric analyses were
carried out on a Beckman DU 800 UV/Vis spectrophotometer. Mass
spectrometric analyses were either performed on Shimadzu LC-MS
IT-TOF or Thermo Fisher Finnigan LCQ Classic spectrometer or ob-
tained from the University of Illinois at Urbana–Champaign Mass
Spectrometry Laboratory. All enzymatic reactions were carried out
in low-retention microcentrifuge tubes to prevent the adsorption
of hydrophobic substrates. All enzyme reaction mixtures contained
10% DMSO to solubilize hydrophobic substrates. These conditions
have only a minimal impact on the natural reaction. Recombinant
Figure 3. BnAP is a selective inhibitor of DXP synthase. A) Design of BnAP as a selective
inhibitor of DXP synthase. B) BnAP is a competitive inhibitor of DXP synthase with re-
spect to pyruvate (K
= (10.4 1.3) mm). The concentration of pyruvate was varied (20–
200 mm) at several fixed concentrations of BnAP (0 (
), 15 (
), 30 (
) and 60 (
) mm) and
100 mmd-GAP; C) BnAP is an uncompetitive inhibitor of DXP synthase with respect to d-
= (70 8) mm). The concentration of d-GAP was varied (10–120 mm) at fixed con-
centrations of BnAP (0 (
), 25 (
), 50 (
) and 75 (
) mm) and 200 mm pyruvate; D) BnAP
is a competitive inhibitor of PDH with respect to pyruvate and exhibits selective inhibi-
tion against DXP synthase compared to PDH (K
= (882 78) mm, K
~85). The
concentration of pyruvate was varied (20–200 mm) at several fixed concentrations of
BnAP (0 (
), 0.5 (
), 1 (
) and 2.25 (
)mm). Representative double reciprocal plots are
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 1309 1315 1313
DXP synthase was purified as previously described.
The protein
concentration was determined by using the Bradford assay. Porcine
pyruvate dehydrogenase was obtained from a commercial source;
its specificity activity was determined by the manufacturer. For
chemical synthesis, CH
was distilled over calcium hydride. An-
hydrous acetonitrile was packed in Sure-Seal bottles. All reactions
were carried out under argon. NMR spectra were recorded on
a Varian 500 MHz spectrometer. Reaction progress was monitored
P NMR with triphenylphosphine oxide (TPPO, d = 0 ppm)
dissolved in deuterated benzene as external standard.
chemical shifts are reported relative to tetramethylsilane (TMS, d =
0 ppm) as internal reference. Preparative HPLC was performed on
a Beckman Gold Noveau system with a Varian Dynamax 250
21.4 mm Microsorb C18 column.
HPLC analysis of DXP synthase-catalyzed CN bond formation
and product characterization: Reaction mixtures containing
HEPES (100 mm, pH 8.0, 2 mm MgCl
,5mm NaCl), ThDP (1 mm),
BSA (1 mg mL
), pyruvate (10–20 mm), DMSO (10%, v/v) and ni-
troso substrate (0.5–5 mm) were preincubated at 378C for 5 min.
Reactions were initiated with enzyme (1–5 mm). Aliquots of the
enzymatic mixture were removed at various time intervals and
quenched in an equal volume of cold methanol. Quenched mix-
tures were incubated on ice for 20 min. Precipitated proteins were
removed by centrifugation, and the supernatant was analyzed by
HPLC with UV detection under the following conditions: flow
rate=3 mLmin
, solvent A: NH
OAc (100 mm, pH 4.6), solvent B:
acetonitrile, method: 0–100 % B over 10 min. The products were
extracted from the supernatant with ethyl acetate (3). The com-
bined organic extracts were concentrated, and the resulting sam-
ples were dissolved in MeOH and resubjected to HPLC analysis to
confirm that product degradation had not taken place during the
extraction procedure. Products were subsequently characterized by
mass spectrometry.
Determination of kinetic parameters for nitroso substrates: Re-
action mixtures containing HEPES (100 m m, pH 8.0, 2 mm MgCl
5mm NaCl), ThDP (1 mm), BSA (1 mgmL
), pyruvate (10–20 mm),
DMSO (10 %, v/v), and nitroso substrate (10–300 mm) were preincu-
bated at 378C for 5 min. Enzymatic reactions were initiated by ad-
dition of DXP synthase (0.5–2 mm; or 0.1 units per mL PDH) and
monitored spectrophotometrically by measuring the rate of disap-
pearance of the nitroso substrate at its corresponding l
. Sub-
strate concentration as a function of time was determined from
absorbance values by using Beer’s Law. Initial reaction rates were
determined from the linear range of the reaction progress curve,
usually within 1–3 min. Data analysis to determine k
and K
each alternative substrate was carried out by using GraFit version 7
from Erithacus Software.
Evaluation of nitroso substrates as inhibitors of DXP formation:
Reaction mixtures containing HEPES (100 mm, pH 8.0, 2 mm MgCl
5mm NaCl), ThDP (1 mm), BSA (1 mgmL
), DMSO (10%, v/v), d-
GAP (30 mm), pyruvate (80 mm), and various concentrations of ni-
troso inhibitor were preincubated at 37 8C for 5 min. Enzyme reac-
tions were initiated by addition of DXP synthase (0.1 mm). Aliquots
(150 mL) of the enzymatic mixture were removed between 0.5 and
3 min and quenched in ice-cold methanol (150 mL). Precipitated
protein was removed by centrifugation, and the supernatant was
diluted in an equal volume of water. The nitroso substrate was re-
moved by extraction into acetonitrile (3) by using a previously
described freeze-extraction technique.
The aqueous layer main-
tained a constant ratio of d-GAP and DXP during the extraction,
and was subjected to derivatization conditions to produce the cor-
responding hydrazones with a fivefold excess of 2,4-dinitrophenyl-
for 20 min to ensure complete derivatization of sub-
strates and product at low concentration. The derivatization mix-
tures were analyzed by HPLC as previously described.
To deter-
mine the initial reaction rates in the presence of various inhibitor
concentrations, the d,l-GAP and DXP hydrazone HPLC peak areas
were measured, and the product concentration was determined as
a percentage of total peak area and plotted against reaction time.
Initial rates GraFit version 7 from Erithacus Software was used to
generate IC
Calculating the active site volume: Coordinates for the ThDP-de-
pendent enzymes, Deinococcus radiodurans DXS (PDB ID: 2O1X),
human PDHE1p (PDB ID: 3EXE),
and transketolase (PDB ID:
were structurally aligned in Coot
by using LSQ Super-
pose and residue ranges A:151–164 (2O1X), E:164–177 (3EXE), and
A:152–165 (3MOS). The choice of residues was based on their
close proximity to ThDP in order to maximize a similar orientation
of the active site region of interest. The RMS deviation, calculated
with VMD
between residues lining the ThDP binding site, was
1.54 (2O1X/3EXE) or 1.01 (2O1X/3MOS) for 16 C
atoms. The biological assembly of transketolase (3MOS) was deter-
mined by using the PISA
web server. Aligned structures were up-
loaded to the Pocket-Finder
web server to determine the vol-
umes of the active site pockets. Cofactors ThDP or ThDP+metal
ions were treated as part of the protein, and all other molecules
were discarded for the purpose of defining the protein surface for
pocket detection. Pocket-Finder reported volumes and generated
space-filling models for the active site pocket in each structure cor-
responding to the pocket adjacent to TDP in chain A of 2O1X. An
overlay of the mesh representations with respect to the active site
cofactor and metal ion was rendered in PyMOL (The PyMOL Molec-
ular Graphics System, Version 1.5.0, Schrçdinger, LLC).
Active site pocket hydrophobicity calculations with fpocket:
(Table S1) was run to detect and analyze pockets in DXP
synthase (2O1X), PDH (3EXE) and TK (3MOS). The complete coordi-
nate files for DXP synthase and PDH, and the biological assembly
for TK, were used as inputs for fpocket. The default cofactor list for
fpocket was modified to include TDP and TPP prior to program
compilation so that the ThDP cofactor would be treated as a part
of the protein as opposed to a removable ligand. The pockets cor-
responding to the active sites used for the volume calculations
with Pocket Finder were determined visually, and the parameters
were recorded.
Synthesis of BnAP (Figure S16): Benzylacetylphosphonate was
prepared starting from phosphorus trichloride in a similar manner
to butylacetylphosphonate,
by using standard procedures. Tri-
benzyl phosphite was generated from benzyl alcohol, diisopropyle-
thylamine, and phosphorous trichloride according to Saady et al.
The spectral properties of the compound were identical to pub-
lished values. For the preparation of benzylacetylphosphonate
(BnAP), a flame-dried flask, cooled under argon, was charged with
acetyl chloride (0.32 mL, 4.5 mmol). Tribenzyl phosphite (0.46 g,
1.3 mmol) was dissolved in anhydrous CH
(13 mL), and the re-
sulting mixture was added dropwise to acetyl chloride. The prog-
ress of the reaction was monitored through
P NMR spectroscopy,
and complete conversion of tribenzyl phosphite (d = 113 ppm) to
dibenzylacetyl-phosphonate (d =26 ppm) was observed within
1 h. Volatiles were removed in vacuo, and the crude material was
used without further purification. Dibenzylacetylphosphonate was
dissolved in anhydrous acetonitrile (2.2 mL), and lithium bromide
(0.17 g, 0.95 mmol) was added in one portion. The reaction mixture
was heated to 508C for ~4 h. The lithium salt of benzylacetyl-
phosphonate precipitated from solution and was removed by
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 1309 1315 1314
filtration. The filter cake was washed successively with cold aceto-
nitrile and diethyl ether (20 mL portions of). The crude product
was purified by reversed-phase preparative HPLC. Flow rate =
10 mL min
; solvent A: HNEt
OAc (50 mm, pH 6.0), solvent B:
methanol, method 5–80% B over 75 min. The purity of fractions
was determined by analytical RP-HPLC. Flow rate=3 mLmin
; sol-
vent A: HNEt
OAc (50 mm, pH 6.0), solvent B: methanol, method 5–
80% B over 12 min. Combined fractions were lyophilized to yield
0.0975 g BnAP as the triethylammonium salt (24 % over two steps).
O): d= 1.20 (t, 9H), 2.31 (d, 3H), 3.11 (m, 6H), 4.91 (d,
2H), 7.35 ppm (m, 5 H);
O): d = 27.43 ppm (s); HRMS
(ESI): m/z calcd for C
P (triethylammonium salt): 316.1678
, found: 316.1673.
Inhibition of DXP synthase by BnAP: In order to evaluate the in-
hibitory activity of BnAP against DXP synthase, a continuous spec-
trophotometric coupled assay was used to measure formation of
DXP by monitoring IspC consumption of NADPH (340 nm).
synthase reaction mixtures (previously described) including BnAP
(15, 30, and 60 mm), IspC (1 mm), and NADPH (100 mm) were prein-
cubated at 378C for 5 min. Initial rates were measured after the re-
action had been initiated by the addition of DXP synthase. Inhibi-
tion of the coupling enzyme (IspC) by BnAP was not observed at
up to 1.5 mm. Experiments were performed in triplicate. Double re-
ciprocal analysis of the data was carried out by using GraFit ver-
sion 7 from Erithacus Software.
Inhibition of PDH by BnAP: Pyruvate dehydrogenase activity was
measured spectrophotometrically as previously reported
monitoring the absorbance changes at 340 nm caused by the re-
duction of NAD
by PDH. Reaction mixtures contained HEPES
(100 mm, pH 8.0), BSA (1 mgmL
), ThDP (0.2 mm), coenzyme A
(0.1 mm), MgCl
(1 mm), cysteine (2 mm), and tris(2-carboxyethyl)-
phosphine (TCEP; 0.3 mm). The reaction was initiated with enzyme
(0.01 units mL
), and activity was monitored at 308C. For inhibition
studies, reaction mixtures (described above) including BnAP (0.5, 1,
2.25 mm) were preincubated at 30 8C for 5 min. Initial rates were
measured immediately after reactions had been initiated by the
addition of PDH (0.01 units mL
). Double reciprocal analysis of the
data was carried out by using GraFit version 7 from Erithacus Soft-
We gratefully acknowledge Katie Heflin for her efforts in the opti-
mization of the HPLC assay used for the inhibition studies. Kip
Bitok is acknowledged for synthesizing tribenzyl phosphite. This
work was supported by funding from The Johns Hopkins Malaria
Research Institute Pilot Grant (F.M.M. and C.F.M.), and the Nation-
al Institutes of Health (GM084998 for C.F.M., F.M.M., R.J.V.;
T32M08018901 for F.M.M., R.J.V., L.B., and AI094967 for F.M.M.)
This work was partially funded through The Bloomberg Family
Foundation (L.B. and J.B.).
Keywords: biosynthesis · DXP synthase · enzyme inhibitors ·
isoprenoids · kinetics · substrate specificity
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Received: March 27, 2013
Published online on July 3, 2013
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 1309 1315 1315
  • [Show abstract] [Hide abstract] ABSTRACT: The unique methylerythritol phosphate pathway for isoprenoid biosynthesis is essential in most bacterial pathogens. The first enzyme in this pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase, catalyzes a distinct thiamin diphosphate (ThDP)-dependent reaction to form DXP from D-glyceraldehyde 3-phosphate (D-GAP) and pyruvate and represents a potential anti-infective drug target. We have previously demonstrated that the unnatural bisubstrate analog, butylacetylphosphonate (BAP), exhibits selective inhibition of Escherichia coli DXP synthase over mammalian ThDP-dependent enzymes. Here, we report the selective inhibition by BAP against recombinant DXP synthase homologs from Mycobacterium tuberculosis, Yersinia pestis and Salmonella enterica. We also demonstrate antimicrobial activity of BAP against both Gram-negative and Gram-positive strains (including E. coli, S. enterica and Bacillus anthracis), and several clinically isolated pathogens. Our results suggest a mechanism of action involving inhibition of DXP synthase and show that BAP acts synergistically with established antimicrobial agents, highlighting a potential strategy to combat emerging resistance in bacterial pathogens.The Journal of Antibiotics advance online publication, 30 October 2013; doi:10.1038/ja.2013.105.
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