Inhibition of IspH, a [4Fe-4S]2+ enzyme involved in the biosynthesis of isoprenoids via the methylerythritol phosphate pathway

Article abstract of DOI:10.1021/ja309557s

The MEP pathway, which is absent in animals but present in most pathogenic bacteria, in the parasite responsible for malaria and in plant plastids, is a target for the development of antimicrobial drugs. IspH, an oxygen-sensitive [4Fe-4S] enzyme, catalyzes the last step of this pathway and converts (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A crucial step in the mechanism of this enzyme is the binding of the C4 hydroxyl of HMBPP to the unique fourth iron site in the [4Fe-4S]²⁺ moiety. Here, we report the synthesis and the kinetic investigations of two new extremely potent inhibitors of E. coli IspH where the OH group of HMBPP is replaced by an amino and a thiol group. (E)-4-Mercapto-3-methylbut-2-en-1-yl diphosphate is a reversible tight-binding inhibitor of IspH with K_i = 20 ± 2 nM. A detailed kinetic analysis revealed that (E)-4-amino-3-methylbut-2-en-1-yl diphosphate is a reversible slow-binding inhibitor of IspH with K_i = 54 ± 19 nM. The slow binding behavior of this inhibitor is best described by a one-step mechanism with the slow step consisting of the formation of the enzyme-inhibitor (EI) complex.

Full text of DOI:10.1021/ja309557s

Article
pubs.acs.org/JACS
Inhibition of IspH, a [4Fe−4S]2+ Enzyme Involved in the Biosynthesis
of Isoprenoids via the Methylerythritol Phosphate Pathway
Karnjapan Janthawornpong,†,∥ Sergiy Krasutsky,‡,§,∥ Philippe Chaignon,† Michel Rohmer,†
C. Dale Poulter,*,‡ and Myriam Seemann*,†
†Universite
́
de Strasbourg, CNRS UMR 7177, Institut Le Bel, 4 rue Blaise Pascal, CS 90032, 67081 Strasbourg Cedex, France
‡Department of Chemistry, University of Utah, 315 South 1400 East Rm 2020, Salt Lake City, Utah 84112, United States
S
* Supporting Information
ABSTRACT: The MEP pathway, which is absent in animals
but present in most pathogenic bacteria, in the parasite
responsible for malaria and in plant plastids, is a target for the
development of antimicrobial drugs. IspH, an oxygen-sensitive
[4Fe−4S] enzyme, catalyzes the last step of this pathway and
converts (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate
(HMBPP) into the two isoprenoid precursors: isopentenyl
diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A crucial step in the mechanism of this enzyme is the binding of
the C4 hydroxyl of HMBPP to the unique fourth iron site in the [4Fe−4S]2+ moiety. Here, we report the synthesis and the
kinetic investigations of two new extremely potent inhibitors of E. coli IspH where the OH group of HMBPP is replaced by an
amino and a thiol group. (E)-4-Mercapto-3-methylbut-2-en-1-yl diphosphate is a reversible tight-binding inhibitor of IspH with
Ki = 20 2 nM. A detailed kinetic analysis revealed that (E)-4-amino-3-methylbut-2-en-1-yl diphosphate is a reversible slow-
binding inhibitor of IspH with Ki = 54 19 nM. The slow binding behavior of this inhibitor is best described by a one-step
mechanism with the slow step consisting of the formation of the enzyme−inhibitor (EI) complex.
Scheme 1. Methylerythritol Phosphate (MEP) Pathway
INTRODUCTION
■
Isoprenoid compounds constitute the most chemically diverse
family of natural products and are found in all living organisms.
The carbon skeletons of isoprenoid molecules are derived from
polyisoprenoid chains formed by the repeated additions of
isopentenyl diphosphate (IPP, 1) to dimethylallyl diphosphate
(DMAPP, 2). These two building blocks are synthesized by
two different routes: the well-established mevalonate pathway1
known since the late 1950s and the more recently discovered
methylerythritol phosphate (MEP) pathway.2,3 In this latter
pathway, IPP and DMAPP are carbohydrate derivatives
synthesized from pyruvate (3) and D-glyceraldehyde phosphate
(4) via 1-deoxy-D-xylulose 5-phosphate (5), 2-C-methyl-D-
erythritol 4-phosphate (6), 4-diphosphocytidyl-2-C-methyl-D-
erythritol (7), 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-
phosphate (8), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate
(9), and (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate
(HMBPP, 10) (Scheme 1).
The MEP pathway is found in most bacteria, including
Mycobacterium tuberculosis responsible for tuberculosis, in the
malaria parasite Plasmodium falciparum, and in the chloroplasts
of plants. The MEP pathway is absent in humans and therefore
presents an appealing target for the development of new
antimicrobials and for herbicides because it provides essential
elements for the photosynthetic apparatus.4,5 Fosmidomycin
inhibits the second enzyme of the pathway, deoxyxylulose
phosphate reductoisomerase (DXR), and is effective alone6 or
in combination with clindamycin7 or artesunate8 for the
treatment of uncomplicated malaria in humans. To date,
fosmidomycin is the only inhibitor of the MEP pathway that is
being investigated clinically. Our interest in the MEP pathway
Received: September 27, 2012
Published: January 14, 2013
© 2013 American Chemical Society
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as a target for new therapeutic agents led us to the design of
new chemical tools to study the reactions catalyzed by IspH,
the last enzyme of the MEP pathway.
Scheme 3. Synthesis of TMBPP (11) and AMBPP (12)
IspH contains an oxygen-sensitive [4Fe−4S]2+ iron−sulfur
center and catalyzes the conversion of HMBPP (10) to a
mixture of IPP (1) and DMAPP (2) (for reviews, see refs 9
and 10).
The [4Fe−4S]2+ cluster in E. coli IspH was identified by EPR
spectroscopy after reconstitution of the enzyme with FeCl3,
Na2S, and DTT in an oxygen-free atmosphere.11 This result
was confirmed by field-dependent Mossbauer spectroscopy,
̈
indicating that IspH contains an unusual [4Fe−4S]2+ cluster in
vivo with one iron atom linked to three inorganic sulfur atoms
from the cluster and to two or three non-sulfur ligands (O and/
or N).12 The first X-ray structures reported for IspH from
Aquifex aeolicus13 and Escherichia coli14 contained a [3Fe−4S]
cluster and, hence, were incomplete. A structure of E. coli IspH
with an intact [4Fe−4S] cluster was only obtained as a complex
with the substrate HMBPP.15
The mechanism catalyzed by IspH requires (i) removal of a
hydroxyl group, (ii) transfer of two electrons from the [4Fe−
4S] cluster, and (iii) the protonation of an intermediate allylic
anion.16 Mossbauer spectroscopy12 and an X-ray structure of
̈
the E. coli IspH·HMBPP complex15 provided the first evidence
for binding of the OH group in HMBPP to the unique fourth
iron site of the [4Fe−4S]2+ cluster of IspH. EPR and ENDOR
spectroscopy performed on a reduced inactive IspH mutant17
and X-ray irradiation of IspH·HMBPP complex crystal15
support the formation of organometallic intermediates.
We synthesized two HMBPP analogues where the OH
group was replaced by an amino or a thiol group known to
coordinate iron atoms to further study the reaction catalyzed by
IspH (Scheme 2). These moieties are known to coordinate to
We initially attempted to introduce the diphosphate moiety by
the procedure of Davisson et al.20 but were unable to prepare
the intermediate chloride from 18 and instead resorted to the
less efficient Cramer phosphorylation.21 Mixed disulfide 19 was
stored at −80 °C, and TMBPP was prepared immediately
before use by treatment with DTT.
AMBPP was prepared by displacement of the hydroxyl group
in 16 with phthalimide, followed by treatment with hydrazine
to give amine 20. The amine was converted to the
corresponding Fmoc carbamate, and the THP group was
removed to give alcohol 21. The alcohol was converted to the
corresponding diphosphate as described for TMBPP, and the
Fmoc group was removed with piperidine to give AMBPP.
IspH. IspH was produced from E. coli strain M15[pREP4,
pQE30-IspH] as a His6-tagged protein and was purified to
homogeneity on a Ni2+-resin affinity column in a glovebox with
a nitrogen atmosphere containing less than 2 ppm oxygen. The
UV/visible spectrum of the protein, the iron and sulfur content,
Scheme 2. Structures of the Thiol (TMBPP, 11) and Amino
(AMBPP, 12) Analogues of HMBPP
and Mossbauer spectra were identical to those previously
̈
reported12 and confirmed that the protein contained an intact
[4Fe−4S]2+ cluster. IspH catalyzed the reduction of HMBPP
to a mixture of IPP and DMAPP (Scheme 1). This reaction
was coupled to a system that reduced the oxidized [4Fe−4S]2+
cluster. In E. coli reduction is facilitated by the natural
flavodoxin/flavodoxin reductase/NADPH system.11,22 In vitro,
reduction can also be performed chemically with the semi-
quinone radical of 5-deazaflavin,11,22 dithionite (DT) reduced
methylviologen (MV), or other dithionite reduced redox
mediators.23 Using a spectroscopic assay based on the change
in NADPH absorbance at 340 nm and optimal concentrations
of NADPH, flavodoxin reductase (FpR1), and flavodoxine
(FldA) in 50 mM Tris HCl buffer, pH 8, IspH activity was
approximately 800 nmol min−1 mg−1, in accordance with
previously reported values.12,24
iron atoms and are poor leaving groups relative to hydroxyl.
The binding mechanisms by which these molecules inhibit E.
coli IspH were investigated by kinetic studies.
RESULTS AND DISCUSSION
■
Synthesis of TMBPP and AMBPP. The divergent
synthesis of TMBPP and AMBPP from THP-protected E-2-
methyl-2-butene-1,4-diol (16) is outlined in Scheme 3. Diethyl
L-tartrate (13) was cleaved with periodate, and the resulting
aldehyde was trapped in situ with the 2-(triphenylphosphor-
anylidene) ylid of propanal,18,19 followed by reduction with
NaBH4 to give hydroxy ester 14. After protection of the alcohol
as a tert-butyldimethylsilyl ether, the ester moiety was reduced
with DIBAL. The resulting alcohol was protected as a THP
ether, and the tert-butyldimethylsilyl group removed by
treatment with fluoride to give 16. TMBPP was synthesized
from 16 by converting the hydroxyl group to the corresponding
chloride, followed by treatment with potassium thioacetate. To
circumvent problems with oxidation of the thiol moiety during
the remaining steps, the thioacetate was converted to the mixed
2-pyridyl disulfide and the THP group was removed to give 18.
TMBPP and AMBPP Are Not Substrates for IspH. IspH
was assayed for turnover under anaerobic conditions by
monitoring the absorbance at 340 nm for incubations
containing 200 μM HMBPP, TMBPP, or AMBPP (as a
control). Figure 1 shows that IspH was fully active in the
presence of HMBPP. Although the rate of NADPH
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conditions as described in Table 1 (conditions b). According to
these studies, 3-butynyl diphosphate was the best inhibitor of
IspH with IC50 = 450 nM for the Aquifex aeolicus enzyme. The
respective IC50 values of 210 and 150 nM for TMBPP and
AMBPP (Table 1, conditions b) with E. coli IspH indicate that
the compounds are potent inhibitors.
Steady-state inhibition studies of IspH with TMBPP were
performed using the biological reducing system NADPH/
FpR1/FldA and [3-14C]HMBPP. The results show that
TMBPP is a competitive inhibitor of IspH with Ki = 20
2
nM (Figure S1). Using a centrifugal filter (Microcon Y-10),
TMBPP was removed from the inhibited IspH·TMBPP
complex by several centrifugation−dilution cycles during
which the enzyme regained its activity, confirming that
TMBPP is a reversible tight-binding inhibitor.
Preliminary experiments were performed to check the ability
of AMBPP to inhibit IspH using NADPH/FpR1/FldA as a
reducing system by monitoring the decrease of absorbance at
340 nm. The progress curves obtained when the reaction was
initiated by addition of HMBPP or by IspH were different
(Figure 2). Upon initiation by addition of IspH, the progress
Figure 1. Decrease of the absorbance of NADPH at 340 nm in the
IspH assay using HMBPP (dark blue), AMBPP (pink), or TMBPP
(green) as substrates or no substrate (cyan). Conditions: NADPH (2.4
mM), FldA (41 μM), FpR1 (17 μM), and IspH (0.5 μM) in 50 mM
Tris HCl buffer, pH 8 (cyan); 200 μM HMBPP (dark blue); 200 μM
AMBPP (pink); 200 μM TMBPP (green). Preincubation for 3 min.
IspH was added with a gastight syringe to initiate the reaction. Samples
were prepared in a glovebox.
consumption was twice the rate of NADPH degradation in the
presence of TMBPP or AMBPP in a control assay containing
no substrate with identical concentrations of cofactors and
enzyme during the first 2 min, no differences were seen
afterward, suggesting that the thiol and amino analogues are not
substrates for IspH.
Inhibition of IspH by TMBPP and AMBPP. Preliminary
experiments were conducted to determine IC50 values for
TMBPP and AMBPP using two different IspH assays
performed under anaerobic conditions. In the first assay,
activity was monitored using [3-14C]HMBPP and the biological
reducing system NADPH/FpR1/FldA. The products were
quantified by liquid scintillation spectrometry upon dephos-
phorylation of IPP and DMAPP with alkaline phosphatase,
releasing isopentenol and dimethylallyl alcohol, followed by
selective heptane extraction of the monoalcohols.11 In the
second assay, IspH activity was determined using dithionite-
reduced methylviologen (DT-reduced MV) as the reducing
agent and by monitoring the decrease of the absorbance of
reduced methylviologen at 732 nm.17,25 As shown in Table 1,
TMBPP and AMBPP are good inhibitors of IspH with IC50
values below 0.5 μM. A series of alkynes26 and pyridine
diphosphates27 were previously tested as IspH inhibitors using
DT-reduced MV as reducing system and the same experimental
Figure 2. Inhibition of IspH by AMBPP (10 μM) in the presence of
HMBPP (200 μM), NADPH (3 mM), FldA (41 μM), FpR1 (17 μM),
IspH (0.5 μM) in 50 mM Tris HCl buffer, pH 8, at 37 °C.
Preincubation for 10 min with HMBPP (a) or IspH (b). Samples were
prepared in a glovebox, and additions were performed with a gastight
syringe.
curve displayed a marked curvature during the first 3−5 min,
followed by linear rate of turnover. However, when the enzyme
was first incubated with AMBPP before addition of HMBPP,
the progress curve was linear. The linear regions of both
progress curves (a and b in Figure 2) had the same slope. This
behavior is typical for a reversible slow-binding inhibitor.28,29
The reversibility of the inhibition was confirmed by removing
the inhibitor from the IspH·AMBPP complex as described
above for TMBPP.
Table 1. IC50 Values for TMBPP and AMBPP with E. coli
IspH
Rates were measured by the decrease in NADPH absorbance
at 340 nm for varied AMBPP concentrations at a fixed
concentration of HMBPP. Because of limitations of working in
an anaerobic environment, approximately 30 s were needed to
add enzyme and mix the sample. Accordingly, 30 s were added
to the time of each run. Progress curves, for formation of the
products in the presence of different AMBPP concentrations
(Figure 3), were generated and fitted to eq 1 (see materials and
methods).
Several mechanisms have been proposed for reversible
competitive slow binding inhibition.28−30 The two widely
accepted models involve slow addition of the inhibitor (I) to
the enzyme to form an E·I complex (mechanism A, see
reducing system
TMBPP IC50 (nM)
AMBPP IC50 (nM)
a
NADPH/FpR1/FldA
480 60
210 10
390 20
150 20
b
DT-reduced MV
a
[3-14C]HMBPP (6.2 μCi μmol−1, 9.4 μM), NADPH (6 mM), FldA
(15 μM), FpR1 (5 μM), IspH (1.6 nM), and varying amounts of
inhibitor in 50 mM Tris HCl buffer, pH 8. IspH was added with a
gastight syringe to initiate the reaction. Incubations were under an
b
argon atmosphere. HMBPP (34 μM), MV (2 mM), DT (0.3 mM),
IspH (74 nM), and varying amounts of inhibitor in 50 mM Tris HCl
buffer, pH 8. Preincubation time was 10 min at 37 °C. Substrate was
added with a gastight syringe to initiate the reaction. Samples were
prepared in a glovebox.
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competitive inhibition constant was estimated to be 35 nM
nM.
8
Similar values for Ki were obtained by analysis of data from
the progress curves and the Lineweaver−Burk plot. The
relatively small difference in Ki probably results from
experimental difficulties related to having to handle an unstable
enzyme in a glovebox. The inhibition constant for AMBPP and
the rate of binding to IspH both appear to decrease as pH
increases (Figures S5 and S6), suggesting that AMBPP is a
better inhibitor in the non-protonated amine form. Because of
the instability of IspH, Ki for AMBPP could not be determined
at higher pH’s.
Mechanistic Considerations. The slow binding behavior
of AMBPP may reflect a requirement that the free amine binds
to the enzyme. Mossbauer studies of IspH in the presence of
̈
either TMBPP and AMBPP indicate that the inhibitors
coordinate to the unique non-sulfur tetracoordinated iron in
the [4Fe−4S]2+ cluster of the enzyme through their respective
thiol and amino groups.31 Thus, the slow binding behavior seen
for AMBPP at pH 8, but not for TMBPP, is consistent with
binding of the free amine and thiol, respectively, to the [4Fe−
4S]2+ cluster.
The X-ray structure of the IspH·HMBPP complex,15 data
from Mossbauer spectroscopy,12,31 and evidence obtained in
̈
this work indicate that HMBPP binds to the [4Fe−4S]2+
cluster in IspH. Analysis of incorporation of deuteriated
isotopologues of 1-deoxy-D-xylulose into pentalenene during
feeding experiments with Streptomyces avermitilis32 as well as
crystallographic studieas of several IspH mutants in complex
with the substrate33 indicate that the CH2OH group in
HMBPP undergoes a rotation by almost 180° to position the
hydroxyl group to interact with E176. An EPR/ENDOR study
of the dithionite reduced inactive E176A mutant of A. aeolicus
IspH in the presence of HMBPP suggests that a one electron
transfer to the [4Fe−4S]2+ cluster in the IspH·HMBPP
complex results in a rearrangement to give a η2-alkenyl complex
(Scheme 4).17 Nevertheless, the structure of the η2-alkenyl
complex, assigned according to 13C-ENDOR studies of the
E126A IspH mutant incubated with [U-13C]HMBPP, is being
challenged by Duin et al. with respect to the observed 13C
couplings (1.7 and 0.8 MHz) that might be larger.34 Proton
transfer to the hydroxyl group and elimination of water and
electron transfer, either simultaneously or sequentially, could
generate the η3-allyl (π) complex10,15,17,22 (Scheme 4). During
X-ray exposure of the IspH·HMBPP complex, an intermediate
lacking the OH group and coordinated to the apical iron of the
[4Fe−4S] via its double bound was observed, although the
mode of bonding between the hydrocarbon moiety and iron
could not be determined from the crystallographic data.10,15
EPR, 31P, and 2H ENDOR investigations of one-electron
reduced A. aeolicus IspH in the presence of HMBPP provided
evidence for the formation of a new reaction intermediate with
spectroscopic properties similar to those of the [4Fe−4S]3+
found in ferredoxin:thioredoxin reductase.34 This latter
intermediate was recently proposed to be the η3-allyl (π)
complex linked to a [4Fe−4S]3+.35 Subsequently, protonation
of the η3-allyl (π) complex at the si face of C-2 will give IPP,
while protonation at C-4 will give DMAPP (Scheme 4).11,16
Elimination of water from HMBPP is a crucial step in the
formation of the putative η3-allyl (π) complex. While the
reactivity of the metallocyclopropyl η2-alkenyl intermediate
toward elimination should be enhanced by one-electron
reduction, it is not known if transfer of the second electron
Figure 3. (A) Progress curves for inhibition of IspH by AMBPP. The
reaction mixture contained NADPH (2 mM), IspH (0.5 μM),
HMBPP (200 μM), FpR1 (17 μM), FldA (41 μM) in 50 mM Tris
HCl buffer, pH 8, and varying concentrations of AMBPP from 0 to
100 μM. After preincubation for 3 min at 37 °C, IspH was added with
a gastight syringe to initiate the reaction. Samples were prepared in a
glovebox. (B) A plot of kapp versus [AMBPP] obtained by fitting the
progress curves to eq 1.
Experimental Procedures) or rapid formation of E·I followed by
a slow conformational change to form an E·I* complex
(mechanism B, see Experimental Procedures). The two
mechanisms can be distinguished from relationships among
the initial rate (v0), the steady-state rate (vs), and the apparent
first-order rate constant for inhibition (kapp) as a function of
inhibitor concentration.28−30 In mechanism A, formation of E·I
is slow. The initial v0 rate is independent of [I], and 1/vs and
kapp are linear functions of [I]. In mechanism B, the formation
of the E·I complex is fast and undergoes a slow isomerization to
E·I*. 1/v0 and 1/vs are linear functions of [I], and kapp is a
hyperbolic function of [I] with limiting values of k6 and k5 + k6.
A plot of the apparent first-order rate constant (kapp) versus
inhibitor concentration was linear (Figure 3B). The reciprocal
plot of the steady-state rate (1/vs) versus [AMBPP] was linear,
and v0 was independent of [I] (Figures 1S and 2S). Fitting the
experimental data to the equation for mechanism A (eq 2) with
KmHMBPP = 1 μM gave k3 = 4.1 × 104 M−1 s−1, k4 = 2.2 × 10−3
s−1, and Ki = 54 19 nM. Thus, inhibition of IspH appears to
proceed by slow addition of AMBPP to free IspH and not a
conformational change of the enzyme−inhibitor complex. The
competitive inhibition was confirmed using the radioactive
assay and varying concentrations of [3-14C]HMBPP and
AMBPP. IspH and AMBPP were preincubated before the
assays were initiated by the addition of the substrate. Double-
reciprocal plots of velocity versus substrate concentration at
different concentrations of AMBPP are given in Figure S4. The
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Scheme 4. Hypothetical Biogenetic Pathway for the IspH-
Catalyzed Reaction via Organometallic Intermediates
EXPERIMENTAL PROCEDURES
■
(E)-4-Mercapto-3-methyl-but-2-en-1-yl Diphosphate Bis
Ammonium Salt (TMBPP, 11). To (E)-3-methyl-4-(pyridin-2-
yldisulfanyl)-but-2-en-1-yl 1-diphosphate 19 (50 mg, 0.12 mmol) in
100 mM NH4HCO3 (1 mL) was added dithiothreitol (DTT, 55 mg,
0.36 mmol) in 100 mM NH4HCO3 (1 mL). After being stirred for 1 h
at room temperature, the mixture was lyophilized, and the residue was
chromatographed on cellulose with elution by 100 mM
NH4HCO3:iPrOH (2:8 v/v). The fractions were analyzed by silica
TLC (iPrOH:H2O:NH4OH (6:1:3 v/v/v)) and visualized by p-
anisaldehyde. The fractions containing the product were combined,
concentrated under reduced pressure, and then lyophilized to give a
white solid (17 mg, 90%); Rf 0.32 (iPrOH:H2O:NH4OH (6:1:3 v/v/
v)); 1H NMR (D2O) 1.79 (3H, s), 3.18 (2H, s), 4.47 (2H, t, J = 8 Hz),
5.62 (1H, m); 13C NMR (D2O) 14.45, 32.37, 62.64 (d, JC,P = 5 Hz),
121.94 (d, JC,P = 8 Hz), 125.12; 31P NMR (D2O) −9.73 (d, J = 22
Hz), −6.92 (d, J = 22 Hz); HRMS (MALDI) calcd for C5H11O7SP2
[M − H] 276.97062, found 276.97112.
(E)-4-Amino-3-methylbut-2-enyl 1-Diphosphate Bis Ammo-
nium Salt (AMBPP, 12). To (E)-(2-methyl-but-2-enyl)-carbamic acid
9H-fluoren-9-yl methyl ester 4-diphosphate 27 (120 mg, 0.23 μmol)
was added piperidine (5 mL). After being stirred for 1 h at room
temperature, piperidine was removed at reduced pressure, and the
residue was purified by flash chromatography on 230−400 mesh silica
with elution by iPrOH:H2O:NH4OH (6:0.5:2.5 v/v/v). The fractions
were analyzed by silica TLC (iPrOH:H2O:NH4OH (6:1:3 v/v/v)) and
visualized by p-anisaldehyde. The fractions containing the product
were combined, concentrated under reduced pressure, and then
lyophilized to give a white solid (58 mg, 85%); Rf 0.1
(iPrOH:H2O:NH4OH (6:1:3 v/v/v)); 1H NMR (D2O) 1.53 (3H,
s), 3.07 (2H, s), 4.32 (2H, t, J = 8 Hz), 5.41 (1H, m); 13C NMR
(D2O) 14.36, 45.51, 62.17 (d, JC,P = 5 Hz), 125.58 (d, JC,P = 8 Hz),
131.94; 31P NMR (D2O) −9.73 (d, J = 22 Hz), −6.92 (d, J = 22 Hz);
HRMS (MALDI) calcd for C5H12NO7P2 [M − H] 260.0095, found
260.0092.
and elimination events are synchronous or stepwise. The
inhibitors of IspH characterized in this work were designed to
replace the hydroxyl moiety in HMBPP with less reactive
amine and thiol leaving groups. As such, they are good tools to
further investigate the IspH mechanism, and we anticipate that
Purification of IspH. The ispH gene of E. coli was cloned into
pQE-30 (Qiagen) as previously described.11 The resulting pQE30-
IspH plasmid was used to transform E. coli M15 [pREP4] to give E.
coli M15 strain [pREP4, pQE30-IspH]. E. coli M15 [pREP4, pQE30-
IspH] was grown at 30 °C on LB medium (6 × 500 mL) containing
Mossbauer, EPR, and X-ray studies of reduced IspH in complex
̈
with TMBPP and AMBPP might provide further information
about the putative η2-alkenyl complex, especially AMBPP, by
blocking the elimination step with concomitant accumulation of
the η2-alkenyl complex.
ampicillin (100 μg mL−1) and kanamycin (25 μg mL−1) to 0.6 OD600
.
FeCl3 (100 μM) was then added, and protein synthesis was induced
with IPTG (100 μM) for 4 h at 30 °C. Cells were harvested by
centrifugation (7000g, 10 min) and kept at −80 °C until used.
The following steps were carried out in a glovebox (Jacomex BS531
T2) equipped with an oxymeter (ARELCO ARC) and filled with a
nitrogen atmosphere containing less than 2 ppm O2. E. coli M15
[pREP4, pQE30-IspH] cells (11 g) were suspended in 50 mM Tris-
HCl buffer (20 mL, pH 8) and sonicated (6 × 40 s with 1 min
cooling). After centrifugation (15 000g, 15 min), the supernatant was
collected and loaded onto an Ni2+-NTA column (Qiagen, 1.2 cm × 7
cm column), equilibrated with 50 mM Tris-HCl bufer, pH 8. The resin
was first washed with 50 mM Tris-HCl buffer, pH 8, containing 20
mM imidazole solution followed by same buffer containing 30 mM
imidazole (20 mL). The His6-IspH protein was eluted with Tris buffer
(8 mL, pH 8) containing 100 mM imidazole to give IspH (27 mg)
after pooling the most colored and pure fractions (purity >90% as
judged by SDS-polyacrylamide gel electrophoresis). After concen-
tration on Microcon YM-30 (Millipore), the resulting brown-green
protein solution (3 mL, 9 mg. mL−1) was divided in aliquots and
stored in liquid nitrogen. Protein concentration was measured by the
method of Bradford using bovine serum albumin as a standard.38 Iron
was quantified by the method of Fish,39 and sulphide was quantified as
described by Beinert.40
CONCLUSIONS
■
In summary, we report the synthesis of (E)-4-mercapto-3-
methylbut-2-en-1-yl diphosphate (TMBPP, 11) and (E)-4-
amino-3-methylbut-2-enyl 1-diphosphate (AMBPP, 12),36 two
HMBPP analogues, where the OH group is replaced by poorer
thiol and amino leaving groups, respectively. Both compounds
are potent inhibitors of E. coli IspH, an enzyme containing an
oxygen-sensitive [4Fe−4S] cluster that catalyzes the final step
in the MEP route to IPP and DMAPP. A steady-state kinetic
study of inhibition of IspH by TMBPP shows that the molecule
is a potent competitive reversible inhibitor with Ki = 24 nM.
AMBPP is also a reversible competitive inhibitor; however, the
molecule shows slow binding behavior where the slow step is
formation of an IspH·AMBPP complex (Ki = 54 nM). We
suggest that the origin of the slow binding behavior is related to
the fact that the free amino group in AMBPP binds to the
fourth iron site of the [4Fe−4S]2+ cluster of IspH and
deprotonation of the dominant protonated form of AMBPP at
physiological pH is a required step. Accordingly, TMBPP (IC50
= 210 nM) and AMBPP (IC50 = 150 nM) are substantially
more potent IspH inhibitors than the previously reported
alkynyl diphosphates (IC50 = 0.45 μM for 3-butynyl
diphosphate with Aquifex aeolicus IspH)26 and pyridine
phosphates (IC50 = 38 μM for BPH-293 with Aquifex aeolicus
IspH),27 which also bind to the [4Fe−4S] cluster.
Spectrophotometric IspH Assay Using NADPH/FpR1/FldA as
Reducing System. HMBPP was synthesized as previously
described.41 In a typical IspH assay, the solution of the enzyme
purified in an oxygen-free glovebox (0.5 μM finale concentration) was
added through a gastight syringe to a 0.1 cm light path cuvette
prepared in the glovebox and containing HMBPP (200 μM), NADPH
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Article
(2.2 mM), flavodoxin (41 μM), and flavodoxin reductase (17 μM) in
Tris-HCl (50 mM, pH 8) that was previously incubated at 37 °C (250
μL final volume). The reaction was monitored photometrically at 340
nm with a Cary 100 UV/visible spectrophotometer (Varian) kept at 37
°C using a thermostat equipped with a Peltier unit. A similar assay was
used to determine if TMBPP and AMBPP were substrates for IspH.
Inhibition Assays Using [3-14C]HMBPP. [3-14C]HMBPP was
synthesized from [2-14C]methyl-D-erythritol 2,4-cyclodiphosphate by
incubation with IspG using flavodoxin (FldA), flavodoxin reductase
(FpR1), and NADPH as the reductant.42 Assays were performed
either on a Schlenk line under argon or in a glovebox under N2
containing less than 2 ppm O2. In a typical experiment, a IspH solution
(5 μL, 34 nM) was added to samples (95 μL) that contained fixed
concentrations of [3-14C]HMBPP, NADPH, FldA, and FpR1 and
varying amounts of AMBPP or TMBPP in 50 mM Tris-HCl buffer,
pH 8 and were preincubated for 3 min at 37 °C. Incubations were kept
at 37 °C for a defined time before addition of 30% trichloroacetic acid
(TCA, 5 μL). The quenched assays were neutralized with 1 N NaOH
(10 μL), centrifuged, and transferred to Eppendorf tubes. Upon
verification that the pH was 8, 50 mM Tris-HCl buffer (300 μL, pH 8)
containing 5 mM MgCl2 was added followed by E. coli alkaline
phosphatase (5 μL, 3.1 mg mL−1, 18 units per mg protein, Sigma).
The aqueous layers were covered with heptane (800 μL) to prevent
the loss of volatile products. Samples were incubated at 25 °C
overnight. Each sample was extracted with heptane (7 × 600 μL), and
the radioactivity of the combined organic layers was measured by
liquid scintillation spectrometry. A control containing no enzyme was
run in parallel.
control sample containing no inhibitor was run under the same
conditions.
Slow-Binding Inhibition. IspH assays in presence of increasing
amounts of AMBPP (0, 5, 10, 25, 50, 100 μM) were performed using
the spectrophotometric assay in the presence of NADPH/FpR1/FldA.
The reactions were initiated by adding IspH with a gastight syringe to
preincubated mixtures containing NADPH, FpR1, FldA, HMBPP, and
AMBPP in 50 mM Tris-HCl buffer. For each recorded absorbance, the
absorbance of the control containing no substrate was subtracted.
Because addition of IspH and mixing required approximately 30 s, this
amount was added to the times for which absorbance was recorded.
For each AMBPP concentration, the absorbance at t = 0 was estimated
from the y-intercept of the straight lines obtained for the first points
and y = 0. Progress curves for formation of the product were plotted
and fitted with Kaleidagraph according to eq 1.
P(t) = vt + (v0 − v)(1 − e−k xt)x(1/kapp
)
s
s
app
(1)
where P(t) is the concentration of product formed (IPP+DMAPP), v0
and vs the initial and steady state rates, t the time, and kapp the apparent
first-order rate constant.29,28 The dependence of v0, vs, and kapp on the
inhibitor concentration [I] permitted one to define if the mechanism
of inhibition follows mechanism A or B.
The radioactivity of assays performed at various inhibitor
concentrations, after subtraction of the radioactivity of the control,
was plotted as dose−response curves and fit using KaleidaGraph
(Synergy software, version 3.5.1 December 2000) according to the
equation y = 1/(1 + (x/IC50)slope) to determine the IC50 values where
y represents the fraction of inhibition and x the inhibitor
concentration.
Steady-state kinetic constants were determined from assays at
several fixed inhibitor concentrations (15, 30, 50 nM) and varying the
substrate concentrations between 0.5 and 4 μM. For the determination
of the Ki of AMBPP under these conditions, HMBPP was used to
initiate the preincubated reactions. The initial velocities and
concentrations were fitted according to the appropriate model of
inhibition.43
Inhibition Assays Using Dithionite-Reduced Methylviolo-
gen. The activity was measured in a similar manner as first published
in ref 25 by using the same IspH and HMBPP concentrations as in ref
26 but reducing the dithionite concentration to 0.3 mM. Typically,
HMBPP (final concentration 34 μM) was added through a gastight
syringe to a 1 cm light path cuvette prepared in a glovebox and
containing a mixture of sodium dithionite (0.3 mM), methylviologen
(2 mM), IspH (74 nM), and various inhibitor concentrations in 50
mM Tris-HCl buffer pH 8, which was previously incubated for 10 min
at 37 °C. The reaction was monitored from changes in absorbance at
732 nm (ε = 3150 M−1 cm−1) with a Cary 100 UV/visible
spectrophotometer (Varian) at 37 °C using a thermostat with a
Peltier element. Initial velocities at various inhibitors concentrations
were plotted as dose−response curves as described above to determine
the IC50 values.
Reversibility of the Inhibition. The following steps were carried
out in a glovebox under nitrogen containing less than 2 ppm of
oxygen. IspH (5 μM) was incubated at 37 °C for 30 min in the
presence of an excess of inhibitor (2 mM) in 50 mM Tris-HCl buffer
(pH 8, final volume 250 μL). A portion of the mixture (25 μL) was
used to initiate a spectrophotometric assay using NADPH/FpR1/FldA
as reducing system to verify that the enzyme was completely inhibited.
The remaining IspH-inhibitor stock solution was concentrated
approximately 5-fold by centrifugation (Microcon Y-10). Buffer (4
volumes) was then added to the concentrate (1 volume), and the
centrifugation−dilution cycle was repeated two more times. The
activity of the resulting mixture was assayed as described above. A
For mechanism A, kapp is a linear function of [I] as described in eq
2.
kapp = k4{1 + [I]/[Ki(1+[S]/Km)]}
(2)
with an inhibition constant Ki = k4/k3.
As the inhibition of IspH by AMBPP followed mechanism A, the
fitting of the data to eq 2 gave the values for k4, k3, and Ki.
For mechanism B, kapp is a hyperbolic function of [I] with limiting
values of k6 and k5 + k6 as described in eq 3:
kapp = k6{1 + [I]/[Ki*(1 + [S]/Km)]}
/{1 + [I]/[Ki(1 + [S]/Km)]}
(3)
with the initial value for complex formation Ki = k4/k3 and the final
value Ki* = Ki[k6/(k5 + k6)].
ASSOCIATED CONTENT
* Supporting Information
Figures S1−S6, Table S1, synthetic procedures, and NMR
spectra for synthetic intermediates. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
mseemann@unistra.fr; poulter@chemistry.utah.edu
■
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Journal of the American Chemical Society
Article
(20) Davisson, V. J.; Woodside, A. B.; Neal, T. R.; Stremler, K. E.;
Present Address
§AMRI Global, Inc. Twenty-six Corporate Circle, P.O. Box
15098, Albany, New York 12212-5098, United States.
Muehlbacher, M.; Poulter, C. D. J. Org. Chem. 1986, 51, 4768−4779.
(21) Cramer, F.; Bohm, W. Angew. Chem. 1959, 71, 775−780.
̈
(22) Rohdich, F.; Zepeck, F.; Adam, P.; Hecht, S.; Kaiser, J.; Laupitz,
Author Contributions
R.; Grawert, T.; Amslinger, S.; Eisenreich, W.; Bacher, A.; Arigoni, D.
̈
∥These authors contributed equally.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 1586−1591.
(23) Xiao, Y.; Chu, L.; Sanakis, Y.; Liu, P. J. Am. Chem. Soc. 2009,
131, 9931−9933.
Notes
The authors declare no competing financial interest.
(24) Grawert, T.; Kaiser, J.; Zepeck, F.; Laupitz, R.; Hecht, S.;
̈
Amslinger, S.; Schramek, N.; Schleicher, E.; Weber, S.; Haslbeck, M.;
ACKNOWLEDGMENTS
Buchner, J.; Rieder, C.; Arigoni, D.; Bacher, A.; Eisenreich, W.;
̈
■
Rohdich, F. J. Am. Chem. Soc. 2004, 126, 12847−12855.
(25) Altincicek, B.; Duin, E. C.; Reichenberg, A.; Hedderich, R.;
Kollas, A. K.; Hintz, M.; Wagner, S.; Wiesner, J.; Beck, E.; Jomaa, H.
FEBS Lett. 2002, 532, 437−440.
This work was supported by grants of the “Agence Nationale de
la Recherche” (ANR-05-JCJC-0177−01) to M.S., by the Thai
government to K.J., and by National Institutes of Health grant
GM 25521 to C.D.P. This Article is dedicated to Professor Dr.
Wolf-D. Woggon on the occasion of his 70th birthday. We
thank Prof. A. Boronat (University of Barcelona, Spain) and his
group for providing the E. coli strain overexpressing IspH. We
are grateful to M. Parisse for technical assistance.
(26) Wang, K.; Wang, W.; No, J. H.; Zhang, Y.; Zang, Y.; Oldfield, E.
J. Am. Chem. Soc. 2010, 132, 6719−6727.
(27) Wang, W.; Li, J.; Wang, K.; Smirnova, T. I.; Oldfield, E. J. Am.
Chem. Soc. 2011, 133, 6525−6528.
(28) Morrison, J. F. Trends Biochem. Sci. 1982, 7, 102−105.
(29) Morrison, J. F.; Walsh, C. T. Adv. Enzymol. Relat. Areas Mol. Biol.
1988, 61, 201−301.
REFERENCES
(1) Bloch, K. Steroids 1992, 57, 378−383.
■
(30) Sculley, M. J.; Morisson, J. F.; Cleland, W. W. Biochim. Biophys.
Acta 1996, 1298, 78−86.
(2) Rohmer, M. Nat. Prod. Rep. 1999, 16, 565−574.
(3) Eisenreich, W.; Rohdich, F.; Bacher, A. Trends Plant Sci. 2001, 6,
78−84.
(31) Ahrens-Botzong, A.; Janthawornpong, K.; Wolny, J. A.;
Ngouamegne Tambou, E.; Rohmer, M.; Krasutsky, S.; Poulter, C.
D.; Schunemann, V.; Seemann, M. Angew. Chem., Int. Ed. 2011, 50,
̈
(4) Rohmer, M.; Grosdemange-Billiard, C.; Seemann, M.; Tritsch, D.
Curr. Opin. Invest. Drugs 2004, 5, 154−162.
11976−11979.
(32) Citron, C. A.; Brock, N. L.; Rabe, P.; Dickschat, J. S. Angew.
Chem., Int. Ed. 2012, 51, 4053−4057.
(5) Eisenreich, W.; Bacher, A.; Arigoni, D.; Rohdich, F. Cell. Mol. Life
Sci. 2004, 61, 1401−1426.
(33) Span, I.; Grawert, T.; Bacher, A.; Eisenreich, W.; Groll, M. J.
̈
(6) Missinou, M. A.; Borrmann, S.; Schindler, A.; Issifou, S.;
Adegnikz, A. A.; Matsiegui, P. B.; Binder, R.; Lell, B.; Wiesner, J.;
Baranek, T.; Jomaa, H.; Kremsner, P. G. Lancet 2002, 360, 1941−
1942.
(7) Borrmann, S.; Lundgren, I.; Oyakhirome, S.; Impouma, B.;
Matsiegui, P. B.; Adegnikz, A. A.; Issifou, S.; Kun, J. F. J.; Hutchinson,
D.; Wiesner, J.; Jomaa, H.; Kremsner, P. G. Antimicrob. Agents
Chemother. 2006, 50, 2713−2718.
Mol. Biol. 2012, 416, 1−9.
(34) Xu, W.; Lees, N. S.; Hall, D.; Welideniya, D.; Hoffman, B. M.;
Duin, E. C. Biochemistry 2012, 51, 4835−4849.
(35) Wang, W.; Wang, K.; Span, I.; Jauch, J.; Bacher, A.; Groll, M.;
Oldfield, E. J. Am. Chem. Soc. 2012, 134, 11225−11234.
(36) A synthesis of (E)-4-amino-3-methylbut-2-enyl 1-diphosphate
(AMBPP) was reported37 while this manuscript was in the final stages
of preparation.
(8) Borrmann, S.; Adegnikz, A. A.; Moussavou, F.; Oyakhirome, S.;
Esser, G.; Matsiegui, P. B.; Ramharter, M.; Lundgren, I.; Kombila, M.;
Issifou, S.; Hutchinson, D.; Wiesner, J.; Jomaa, H.; Kremsner, P. G.
Antimicrob. Agents Chemother. 2005, 50, 3749−3754.
(9) Seemann, M.; Rohmer, M. C. R. Chimie 2007, 10, 748−755.
(37) Das, D.; Tnimov, Z.; Nguyen, U. T. T.; Thimmaiah, G.; Lo, H.;
Abankwa, D.; Wu, Y.; Goody, R. S.; Waldmann, H.; Alexandrov, K.
ChemBioChem 2012, 13, 674−683.
(38) Bradford, M. M. Anal. Biochem. 1976, 72, 248−254.
(39) Fish, W. W. Methods Enzymol. 1988, 158, 357−364.
(40) Beinert, H. Anal. Biochem. 1983, 131, 373−378.
(41) Wolff, M.; Seemann, M.; Grosdemange-Billiard, C.; Tritsch, D.;
Campos, N.; Rodríguez-Concepcíon, M.; Boronat, A.; Rohmer, M.
Tetrahedron Lett. 2002, 43, 2555−2559.
(42) Seemann, M.; Tse Sum Bui, B.; Wolff, M.; Tritsch, D.; Campos,
N.; Boronat, A.; Marquet, A.; Rohmer, M. Angew. Chem., Int. Ed. 2002,
41, 4337−4339.
(43) Segel, I. H. Enzyme Kinetics: Behavior and Analysis of Rapid
Equilibrium and Steady-State Enzyme Systems; J. Wiley & Sons, Inc.:
New York, 1975.
(10) Grawert, T.; Span, I.; Bacher, A.; Groll, M. Angew. Chem., Int.
̈
Ed. 2010, 49, 8802−8809.
(11) Wolff, M.; Seemann, M.; Tse Sum Bui, B.; Frapart, Y.; Tritsch,
́
D.; Garcia-Estrabot, A.; Rodriguez-Concepcion, M.; Boronat, A.;
Marquet, A.; Rohmer, M. FEBS Lett. 2003, 541, 115−120.
(12) Seemann, M.; Janthawornpong, K.; Schweizer, J.; Bottger, L. H.;
̈
Janoschka, A.; Ahrens-Botzong, A.; Ngouamegne Tambou, E.;
Rotthaus, O.; Trautwein, A. X.; Rohmer, M.; Schunemann, V. J. Am.
Chem. Soc. 2009, 131, 13184−13185.
(13) Rekittke, I.; Wiesner, J.; Rohrich, R.; Demmer, U.; Warkentin,
̈
̈
E.; Xu, W.; Troschke, K.; Hintz, M.; No, J. H.; Duin, E. C.; Oldfield,
E.; Jomaa, H.; Ermler, U. J. Am. Chem. Soc. 2008, 130, 17206−17207.
(14) Grawert, T.; Rohdich, F.; Span, I.; Bacher, A.; Eisenreich, W.;
̈
Eppinger, J.; Groll, M. Angew. Chem., Int. Ed. 2009, 48, 5756−5759.
(15) Grawert, T.; Span, I.; Eisenreich, W.; Rohdich, F.; Eppinger, J.;
̈
Bacher, A.; Groll, M. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 1077−
1081.
(16) Laupitz, R.; Grawert, T.; Rieder, C.; Zepeck, F.; Bacher, A.;
̈
Arigoni, D.; Rohdich, F.; Eisenreich, W. Chem. Biodiversity 2004, 1,
1367−1376.
(17) Wang, W.; Wang, K.; Liu, Y. L.; No, J. H.; Li, J.; Nilges, M. J.;
Oldfield, E. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 4522−4527.
(18) Dunlap, N. K.; Mergo, W.; Jones, J. M.; Carrick, J. D.
Tetrahedron Lett. 2002, 43, 3923−3925.
(19) Outram, H. S.; Raw, S. A.; Taylor, J. K. Tetrahedron Lett. 2002,
43, 6185−6187.
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dx.doi.org/10.1021/ja309557s | J. Am. Chem. Soc. 2013, 135, 1816−1822