Probing ligand-binding pockets of the mevalonate pathway enzymes from Streptococcus pneumoniae

Article abstract of DOI:10.1074/jbc.M109.098350

Diphosphomevalonate (Mev·pp) is the founding member of a new class of potential antibiotics targeting the Streptococcus pneumoniae mevalonate (Mev) pathway. We have synthesized a series of Mev·pp analogues designed to simultaneously block two steps in this pathway, through allosteric inhibition of mevalonate kinase (MK) and, for five of the analogues, by mechanismbased inactivation of diphosphomevalonate decarboxylase (DPM-DC). The analogue series expands the C₃-methyl group of Mev·pp with hydrocarbons of varying size, shape, and chemical and physical properties. Previously, we established the feasibility of a prodrug strategy in which unphosphorylated Mev analogues could be enzymatically converted to the active Mev·pp forms by the endogenous MK and phosphomevalonate kinase.Wenowreport the kinetic parameters for the turnover of non-, mono-, and diphosphorylated analogues as substrates and inhibitors of the three mevalonate pathway enzymes. The inhibition of MK by Mev·pp analogues revealed that the allosteric site is selective for compact, electron-rich C₃-subsitutents. The lack of reactivity of analogues with DPM-DC provided evidence, counter to the existing model, for a decarboxylation transition state that is concerted rather than dissociative. The Mev pathway is composed of three structurally and functionally conserved enzymes that catalyze consecutive steps in a metabolic pathway. The current work reveals that these enzymes exhibit significant differences in specificity toward R-group substitution at C₃ and that these patterns are explained well by changes in the volume of the C₃ R-group-binding pockets of the enzymes.

Full text of DOI:10.1074/jbc.M109.098350

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 27, pp. 20654–20663, July 2, 2010
2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
©
Probing Ligand-binding Pockets of the Mevalonate Pathway
□
S
*
Enzymes from Streptococcus pneumoniae
Received for publication, December 23, 2009, and in revised form, April 2, 2010 Published, JBC Papers in Press, April 19, 2010, DOI 10.1074/jbc.M109.098350
‡1
‡
1
§
¶
§
‡2
Scott T. Lefurgy , Sofia B. Rodriguez , Chan Sun Park , Sean Cahill , Richard B. Silverman , and Thomas S. Leyh
‡
¶
From the Departments of Microbiology & Immunology and Biochemistry, Albert Einstein College of Medicine, Bronx,
New York 10461 and the Department of Chemistry, Department of Biochemistry, Molecular Biology, and Cell Biology,
§
Center for Molecular Innovation and Drug Discovery, and Chemistry of Life Processes Institute, Northwestern University,
Evanston, Illinois 60208-3113
Diphosphomevalonate (Mevꢀpp) is the founding member of a new antibiotic strategies to combat unvaccinated strains, which
new class of potential antibiotics targeting the Streptococcus are rapidly filling the biological niches left by the vaccine (5).
3
pneumoniae mevalonate (Mev) pathway. We have synthesized a
The mevalonate (Mev) pathway is essential for the survival
series of Mevꢀpp analogues designed to simultaneously block of S. pneumoniae in lung and serum (6). The bacterium uses this
two steps in this pathway, through allosteric inhibition of meva- pathway to convert Mev to isopentenyl diphosphate, the “build-
lonate kinase (MK) and, for five of the analogues, by mechanism- ing block” of the isoprenoids: a class of 25,000 unique molecules
based inactivation of diphosphomevalonate decarboxylase having a wide range of biological functions. The pathway con-
(
DPM-DC). The analogue series expands the C -methyl group sists of three GHMP family kinases: Mev kinase (MK), phos-
3
of Mevꢀpp with hydrocarbons of varying size, shape, and chem- phomevalonate kinase (PMK) and diphosphomevalonate
ical and physical properties. Previously, we established the fea- decarboxylase (DPM-DC) (Fig. 1) (7). Mutations that knock out
sibility of a prodrug strategy in which unphosphorylated Mev genes in this pathway kill the organism in vivo, suggesting that
analogues could be enzymatically converted to the active S. pneumoniae cannot obtain the necessary precursors or
Mevꢀpp forms by the endogenous MK and phosphomevalonate downstream products from the host (6). In principle, each of
kinase. We now report the kinetic parameters for the turnover of the three enzymes is an antibiotic target, because inhibition of
non-, mono-, and diphosphorylated analogues as substrates and any of them prevents the production of isopentenyl diphos-
inhibitors of the three mevalonate pathway enzymes. The inhi- phate. We have shown that S. pneumoniae MK is potently (K ϭ
i
bition of MK by Mevꢀpp analogues revealed that the allosteric site 500 nM) allosterically inhibited by diphosphomevalonate
is selective for compact, electron-rich C -subsitutents. The lack of (Mevꢀpp), the third compound in the pathway, whereas the
3
reactivity of analogues with DPM-DC provided evidence, counter human MK homologue is not (8). Thus, the allosteric site pro-
to the existing model, for a decarboxylation transition state that is vides an opportunity to selectively target the bacterium. The S.
concerted rather than dissociative. The Mev pathway is composed pneumoniae MK crystal structure revealed a pore at the subunit
of three structurally and functionally conserved enzymes that cat- interface with excellent charge- and shape-complementarity to
alyze consecutive steps in a metabolic pathway. The current work Mevꢀpp that may be the allosteric site.
reveals that these enzymes exhibit significant differences in speci-
In an effort to enhance the inhibitory properties of Mevꢀpp
ficity toward R-group substitution at C and that these patterns are for use as an antibiotic, we have built a series of ten Mev ana-
3
explained well by changes in the volume of the C R-group-binding logues (9) in which the C -methyl group has been altered to
3
3
pockets of the enzymes.
other hydrocarbon substituents (Table 1). Five of the analogues
have linear and branched alkyl groups at C that act as struc-
3
tural probes for the MK allosteric site and the active sites of the
Streptococcus pneumoniae, the primary cause of bacterial three GHMP kinases. The remaining five analogues resemble
meningitis and pneumonia, kills over 4000 people daily world- the alkyl series but also have the potential to act as mechanism-
wide and disproportionately affects children and the elderly (1, based inhibitors of DPM-DC.
2
). Antibiotic resistance is a major problem in fighting this
In the DPM-DC mechanism, the C -hydroxyl of Mevꢀpp is
3
organism, with multiple-drug resistance rates as high as 95% in phosphorylated by ATP, generating pꢀMevꢀpp, which ionizes,
some regions (3). Although vaccines targeting the 7 or 23 most leaving a carbocation on C ; decarboxylation is thought to fol-
3
prevalent strains have shown success in reducing disease inci- low rapidly (10). Abeles and coworkers have provided evidence
dence in developed countries (4), there is a continual need for
3
The abbreviations used are: Mev, mevalonate; Mevꢀp, phosphomevalonate;
Mevꢀpp, diphosphomevalonate; pꢀMevꢀpp, 3-phospho-5-diphosphom-
*
Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grant AI 068989.
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental text, Fig. S1, Table S1, and Schemes S1–S3.
Both authors contributed equally to this work.
To whom correspondence should be addressed: 1300 Morris Park Ave.,
Bronx, NY 10461. Tel.: 718-430-2858; Fax: 718-430-8711; E-mail:
thomas.leyh@einstein.yu.edu.
evalonate; DPM-DC, diphosphomevalonate decarboxylase; HMBC, hetero-
nuclear multiple-bond connectivity; HSQC, heteronuclear single quantum
coherence; LDH, lactate dehydrogenase; 2-ME, ␤-mercaptoethanol; MK,
Mev kinase; PEP, phosphoenolpyruvate; PK, pyruvate kinase; PMK, phos-
phomevalonate kinase; unit, 1 ␮mol of product formed per minute at a
saturating concentration of one or more substrates; X, the Mev analogue
series; Xꢀp, the phosphomevalonate analogue series; Xꢀpp, the diphos-
phomevalonate analogue series.
□
1
2
2
0654 JOURNAL OF BIOLOGICAL CHEMISTRY
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S. pneumoniae Mevalonate Pathway Enzymes
FIGURE 1. The mevalonate pathway. The conversion of mevalonate to isopentenyl diphosphate occurs in three ATP-dependent steps catalyzed by GHMP
family kinases: MK, mevalonate kinase; PMK, phosphomevalonate kinase; DPM-DC, diphosphomevalonate decarboxylase.
with the mammalian enzyme that a carbocation must form dur- tures given in Table 1. Where appropriate, numbers are
ing turnover by showing that substitution of the C -methyl appended with “ꢀp” or “ꢀpp” to indicate the number of phos-
3
group with a hydrogen or a fluoromethyl group causes the reac- phates on the C -hydroxyl. For example, 6ꢀpp refers to the
5
tion to halt after phosphorylation, implying that the electron- 5Ј-diphosphorylated form of the vinyl analogue (see Table 1). A
donating methyl group is required to stabilize the carbocation group of analogues of a particular phosphorylation state is
inductively (10). To exploit the potential carbocation formation referred to using a boldface X in place of the number (i.e. X, Xꢀp,
for DPM-DC inhibition, we replaced the C -methyl group with or Xꢀpp).
3
substituents that could undergo resonance with the carboca-
tion, thereby setting up an electrophilic site for nucleophilic
attack by the enzyme (Fig. 2). Because the inhibitors are struc-
turally related to Mevꢀpp, we expected that they might also
inhibit MK through binding at the allosteric site. By targeting
two steps in the same pathway, such molecules are expected to
have enhanced antibiotic potency.
Enzymatic Synthesis of Stereochemically Pure Substrates of
the Mevalonate Pathway—A series of (R,S)-mevalonolactone
analogues ((R,S)-X) were synthesized as racemic mixtures as
described (9). Approximately 25 mg of each lactone was treated
with five equivalents of KOH at 37 °C for 1 h to convert it to the
corresponding carboxylate. The solution was then adjusted to
ϩ
pH 7.0 with 1 M HCl and diluted with Hepes/K (50 mM, pH
We now report the chemical and chemoenzymatic synthesis
of the Mevꢀpp analogue series, the kinetic parameters of the
Mev, Mevꢀp, and Mevꢀpp analogues as substrates of their three
respective Mev pathway GHMP kinases, and the inhibition of
MK and DPM-DC by Mevꢀpp analogues, noting their potential
as antibiotics and the substrate-binding features of the enzyme
family that they reveal.
7
7
.0) and MgCl (1.0 mM) to a final concentration of ϳ240 mM in
2
50 ␮l. Concentration of the R-isomer was determined by enzy-
matic assay (see below).
Stereochemically pure mono- and diphosphorylated Mev
analogues ((R)-Xꢀp and (R)-Xꢀpp (X ϭ 1, 3, 6, and 7)) were
enzymatically synthesized from racemic Mev analogues
(
R,S)-X in successive steps catalyzed by MK and PMK. The first
phosphorylation reaction contained S. pneumoniae MK (0.350
M), (R,S)-X (ϳ7.5 mM), ATP (4.5 mM), PK (10 units/ml), PEP
EXPERIMENTAL PROCEDURES
␮
(
General Materials and Methods—Lactate dehydrogenase
ϩ
10 mM), 2-ME (20 mM), MgCl (5.5 mM), and Hepes/K (50
2
(
LDH, rabbit muscle), pyruvate kinase (PK, rabbit muscle), and
mM, pH 8.0) After 72 h at room temperature, 98% of the starting
material had been converted to the (R)-Xꢀp analogue. The sec-
ond phosphorylation was initiated by the addition of PMK (0.28
alkaline phosphatase (calf intestine) were purchased from
Roche Applied Science and treated as described previously (11).
ATP, ADP, NADH, phosphoenolpyruvate (PEP), ␤-mercapto-
ethanol (2-ME), (R,S)-mevalonate (Mev), and triethylamine
␮
M) and PEP (1.0 mM) to the reaction mixture, and this reaction
achieved 99% conversion to (R)-Xꢀpp analogue after ϳ24 h.
Because (R,S)-9 was known to be a slow substrate for S. pneu-
moniae MK, the production of (R)-9ꢀpp was facilitated using a
one-pot reaction that included S. aureus MK, which is
weakly inhibited by Mevꢀpp, and PMK. The reaction was
completed in 36 h. (R)-9ꢀp was synthesized as described
above, but starting from (R)-9. (R)-X compounds (X ϭ 1, 3,
were purchased from Sigma. NaCl, KCl, Hepes, Tris, MgCl ,
2
KOH, glycerol, and Luria-Bertani Miller media were purchased
from Fisher Scientific. Isopropyl-1-thio-␤-D-galactopyranoside
was purchased from Labscientific, Inc. Q-Sepharose Fast Flow
ion-exchange, Chelating Sepharose Fast Flow, and Glutathi-
one-Sepharose 4 Fast Flow resins were purchased from Amer-
sham Biosciences. Anion-exchange resin AG MP-1 was pur-
chased from Bio-Rad. YM-10 membranes were purchased from
Millipore. Competent Escherichia coli BL21(DE3) was pur-
chased from Novagen. Racemic mevalonolactone analogues
6
, 7, and 9) were generated by removing the pyrophosphoryl
moiety from two-thirds of the purified (see below) (R)-Xꢀpp
using alkaline phosphatase. The reaction mixtures con-
tained: alkaline phosphatase (2.5 units/ml), (R)-Xꢀpp (2.4
mM), Tris/HCl (50 mM, pH 8.5) at 37 °C (98% conversion to
product was reached in 1 h). The (R)-X analogues were found
to lactonize upon purification (see below) and were saponi-
(
(R,S)-X), Streptococcus pneumoniae MK, PMK, DPM-DC, and
Staphylococcus aureus MK were prepared as described previ-
¨
ously (9, 11). Chromatography was carried out using an AKTA
fast-protein liquid chromatography system (Amersham Bio-
sciences). Spectrophotometric measurements were carried out fied to the carboxylates as described above.
using a Cary 100 or 400 UV-visible spectrophotometer (Varian,
Reaction progress and analogue concentration were moni-
Inc.). Fluorometric measurements were carried out using a tored by enzymatic assay (12). MK, PMK, and DPM-DC pro-
Cary Eclipse fluorometer (Varian, Inc.).
duce ADP in the presence of their respective substrates; ADP
Chemical Nomenclature—In the description of kinetic formation was measured spectrophotometrically at 339 nm by
experiments, the substrate analogues (in free acid form) are stoichiometrically coupling (1:1) ADP production to NADH
referred to by boldface numbers that correspond to the struc- oxidation using the well established PK/LDH-coupled assay
JULY 2, 2010•VOLUME 285•NUMBER 27
JOURNAL OF BIOLOGICAL CHEMISTRY 20655

S. pneumoniae Mevalonate Pathway Enzymes
logues eluted at 0.2, 0.285, and 0.33
M KCl, respectively, and contained
Ͻ1% nucleotide. Each purified ana-
logue was desalted by loading onto
an anion-exchange resin (Q-Sepha-
rose Fast Flow, 5 ml) and washing
(
10 mM) and eluting (1.2 M) with tri-
ethylamine/HCO (pH 7.0). Excess
3
triethylamine was removed by
rotary evaporation, washing with
1
00% methanol (25 ml, six times)
and ultrapure water (25 ml, three
times). Solution pH was adjusted to
7
.0 with HCl, and the concentration
of the analogue was determined by
enzymatic assay.
Determination of Initial Rate
Constants—Initial rate constants of
MK, PMK, and DPM-DC reactions
with substrate analogues were
determined by a progress curve
method. Complete MK reaction
progress curves were obtained by
monitoring absorbance at 398 nm.
Products were removed by includ-
ing PMK and DPM-DC in the reac-
tion mixture to prevent possible
product inhibition; therefore, three
molar equivalents of NADH are oxi-
dized per MK substrate. (R)-1, -3,
-
6, -7, and -9 were previously shown
to be substrates for MK (9). The
concentration of acceptor (Mev or
analogue) and MK varied according
to the kinetic constants of the
FIGURE 2. DPM-DC inactivation hypothesis. The dissociative model of Mevꢀpp decarboxylation (shown for
the 9ꢀpp analogue) begins with phosphorylation of the C -OH by ATP. Phosphate then ionizes, leaving a C
3
3
carbocation, which either rapidly decarboxylates to form the double-bonded product (left resonance form) or, acceptor. The conditions were as
in the case of analogues 6ꢀpp through 10ꢀpp, rearranges and is quenched by a nucleophile on the protein
surface (right resonance form), forming a covalent adduct. Opening the cyclopropyl ring of the 9ꢀpp analogue
results in a homoallyl carbocation that is stabilized, relative to the ring-intact carbocation, as a result of the
release of the ϳ27 kcal/mol ring strain (29, 30).
follows: Mev (135 ␮M), MK (100
nM); 1 (400 ␮M), MK (500 nM); 3 (1.0
mM), MK (10 ␮M); 6 (400 ␮M), MK
(
500 nM); 7 (400 ␮M), MK (1.0 ␮M);
system (13). The assay conditions were as follows: S. pneu- and 9 (600 ␮M), MK (1.0 ␮M). The reaction mixture contained:
ϩ
moniae MK, PMK, or DPM-DC (1.0 ␮M), reaction mixture or ATP (5.0 mM, 30ꢀK ), MgCl (6.0 mM), Hepes/K (50 mM, pH
m
2
ϩ
analogue (5 ␮l), ATP (3.0 mM), MgCl (4.0 mM), Hepes/K (50 8.0), KCl (50 mM), PK (5.0 units/ml), LDH (10 units/ml), PEP
2
mM, pH 8.0), KCl (50 mM), PK (6.0 units/ml), LDH (12 units/ (4.0 mM), 2-ME (10 mM), and NADH (7.0 mM, ⌬⑀ ϭ 0.136
3
98
Ϫ1
Ϫ1
ml), PEP (5.0 mM), 2-ME (10 mM), and NADH (200 ␮M, ⌬⑀ ϭ mM cm ); T ϭ 25 Ϯ 2 °C. The concentrations of the cou-
Ϫ1
Ϫ1
6
.22 mM cm ). For each mole of (R,S)-X in the initial reac- pling enzymes PMK and DPM-DC were selected to minimize
tion, 0.5 mol of product was produced, representing a biologi- their contribution to the lag time of the assay (14); consequently
cally relevant (R)-isomer. ADP (2.0 mM) was added at the end of they varied according to the kinetic constants of these enzymes
synthesis reactions to completely convert PEP to pyruvate, toward a given acceptor. The concentrations, given in the for-
facilitating purification of the phosphorylated Mev species.
mat (acceptor, [PMK] (␮M), [DPM-DC] (␮M)), were as follows:
Purification of R-Mevalonate, 5-Monophosphate, and (Mev, 0.15, 0.30), (1, 3.0, 2.0), (3, 15, 15), (6, 1.5, 1.0), (7, 2.0, 3.0),
-Diphosphate Analogues—A reaction mixture containing and (9, 3.0, 2.0). The maximum concentrations of (R)-1, -3, -6,
5
(
R)-X, (R)-Xꢀp, or (R)-Xꢀpp was separated from enzymes by -7, and -9 that could reasonably be achieved in the assay were
passing the solution through a YM-10 membrane. The filtrate too low relative to their K to obtain precise K values; conse-
m
m
was loaded onto an anion-exchange resin (AG MP-1, 35 ml) quently, only V/K values were determined (see Table 1).
ϩ
equilibrated with Hepes/K (10 mM, pH 7.5), washed five times
with equilibration buffer, and eluted using a linear KCl gradient strates for MK; product was not detected under the following
0–1.0 M, 750 ml, 2 ml/min). (R)-X, (R)-Xꢀp, and (R)-Xꢀpp ana- conditions: (R,S)-X (1.0 mM), MK (10 ␮M), PMK (1.0 ␮M),
(R,S)-2, -4, -5, -8, and -10 proved to be extremely poor sub-
(
2
0656 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 27•JULY 2, 2010

S. pneumoniae Mevalonate Pathway Enzymes
DPM-DC (2.0 ␮M), ATP (8.0 mM, 9.5ꢀK ), MgCl (9.0 mM), ting complete reaction progress curves to the integrated
m
2
ϩ
Hepes/K (50 mM, pH 8.0), KCl (50 mM), PK (6.0 units/ml), Michaelis-Menten equation (Equation 1), using a FORTRAN77
LDH (12 units/ml), PEP (4.0 mM), 2-ME (10 mM), and NADH program of Cleland (15) adapted by one of us (S. T. L.) for this
Ϫ1
Ϫ1
(
2.0 mM, ⌬⑀ ϭ 0.61 mM cm ); T ϭ 25 Ϯ 2 °C. No diphos- purpose (available upon request). Prior to analysis, the linear
3
86
phorylated product was detected over a 4-h period. The limit of background ATP hydrolysis was subtracted from the curve, and
Ϫ1
detection of the instrument (⌬A ϭ 0.025 h ) corresponds the data were manually truncated to remove the lag in NADH
386
Ϫ1
to a maximum rate of 0.067 ␮M min
.
oxidation due to coupling enzymes. Absorbance values were
PMK reaction progress curves were measured by monitoring normalized to the minimum absorbance value and converted to
absorbance at 339 nm. The product was removed by including substrate concentrations (S ) using the extinction coefficient of
t
DPM-DC in the reaction mixture to prevent possible product NADH and equivalents of ADP produced in each reaction (see
inhibition; therefore, two molar equivalents of NADH are pro- above). The initial substrate concentration (S ) was taken from
0
duced per PMK substrate. The concentration of analogue and the corrected, normalized absorbance at t ϭ 0.
PMK varied in each experiment: Mevꢀp (100 ␮M), PMK (10 nM);
1
S0
1
ꢀp (600 ␮M), PMK (150 nM); 3ꢀp (600 ␮M), PMK (2.0 ␮M); 6ꢀp
t ϭ
Km ln ϩ ͑S Ϫ S ͒
(Eq. 1)
ͫ
0
t
ͬ
(
(
(
600 ␮M), PMK (75 nM); 7ꢀp (600 ␮M), PMK (100 nM); and 9ꢀp
k
cat͓E͔
S
t
600 ␮M), PMK (500 nM). The reaction mixture contained: ATP
ϩ
Fitted parameters k and K (p ) and associated errors from
4.0 mM, 30ꢀK ), MgCl (5.0 mM), Hepes/K (50 mM, pH 8.0),
cat
m
n
m
2
two or three independent progress curves of each enzyme-sub-
KCl (50 mM), PK (6.0 units/ml), LDH (12 units/ml), PEP (4.0
strate pair were averaged (16). Mean parameter values ( pˆ ) and
mM), 2-ME (10 mM), and NADH (2.0 mM, ⌬⑀
ϭ 0.61
386
Ϫ1
Ϫ1
their associated errors (␴ ) were determined using Equations 2
mM cm ); T ϭ 25 Ϯ 2 °C. The concentration of DPM-DC
varied according to the kinetic constants for a given analogue.
The concentrations, given as (acceptor, [DPM-DC] (␮M)), were
as follows: (Mevꢀp, 0.50), (1, 3.0), (3, 20); (6, 1.5), (7, 1.5), (9, 3.0).
Analogues 2ꢀp, 4ꢀp, 5ꢀp, 8ꢀp, and 10ꢀp could not be produced
enzymatically using MK and were not tested.
pˆ
and 3. Weighting factors (a ) were obtained from Equation 4,
n
2
where the variance (␴ ) is the square of the standard deviation
n
(
␴ ) of the least-squares fitted parameter.
n
pˆ ϭ a p ϩ a p ϩ a p
3 3
(Eq. 2)
(Eq. 3)
1
1
2
2
Complete DPM-DC reaction progress curves for (R)-1ꢀpp,
2
2
2
2
2
2
V͑ pˆ ͒ ϭ a ␴ ϩ a ␴ ϩ a ␴
3
1
1
2
2
3
3
ꢀpp, 6ꢀpp, 7ꢀpp, and 9ꢀpp were measured by the change in
2
fluorescence upon oxidation of NADH (␭ ϭ 339 nm and
Ex
1/␴n
an ϭ
(Eq. 4)
␭
ϭ 466 nm). The amount of NADH oxidized during the
2
2
2
Em
͑
1/␴ ͒ ϩ ͑1/␴ ͒ ϩ ͑1/␴ ͒
1
2
3
incubation was calculated by comparison to a standard curve;
NADH fluorescence gave a linear response up to 10 ␮M. The
S. pneumoniae DPM-DC Homology Model with Mevꢀpp—A
concentration of analogue and DPM-DC varied in each exper- homology model of the S. pneumoniae enzyme was generated
iment: Mevꢀpp (5.0 ␮M), DPM-DC (2.5 nM); 1ꢀpp (3.0 ␮M), in Swiss PDB Viewer (17). from the crystal structure of S. pyo-
DPM-DC (5.0 nM); 3ꢀpp (6.0 ␮M), DPM-DC (30 nM); 6ꢀpp (6.0 genes DPM-DC (70% sequence identity, PDB: 2GS8). The fif-
␮
9
M), DPM-DC (2.5 nM); 7ꢀpp (6.0 ␮M), DPM-DC (15 nM); and teen active-site residues used to position Mevꢀpp in the model
ꢀpp (2.6 ␮M), DPM-DC (30 nM). The reaction mixture con- are completely conserved in the two sequences. Mevꢀpp was
ϩ
tained: ATP (4.0 mM, 55ꢀK ), MgCl (5.0 mM), Hepes/K (50 manually positioned using the criteria of Byres et al. (18), which
m
2
mM, pH 8.0), KCl (50 mM), PK (4.0 units/ml), LDH (8.0 units/ were used to position Mevꢀpp in the apo Trypanosoma brucei
ml), PEP (2.0 mM), 2-ME (10 mM), and NADH (13 ␮M); T ϭ DPM-DC structure (PDB: 2HKE). To facilitate comparison of
2
5 Ϯ 2 °C. (R,S)-2ꢀpp, 4ꢀpp, 5ꢀpp, 8ꢀpp, and 10ꢀpp progress the two structures, the T. brucei structure was aligned with the
curves were acquired by monitoring absorbance at 339 nm to S. pneumoniae homology model, using PyMOL (19). The
accommodate the higher substrate concentrations needed to Mevꢀpp ligand was then positioned using the criteria below to
determine K . The concentration of the (R,S) analogue ((R)- determine placement of the terminal phosphate, the C -car-
m
1
isomer) and DPM-DC varied in each experiment: 2ꢀpp (17 ␮M), boxylate, and the C -hydroxyl. The terminal phosphate of
3
DPM-DC (3.0 ␮M); 4ꢀpp (64 ␮M), DPM-DC (3.2 ␮M); 5ꢀpp (17 Mevꢀpp in the T. brucei model was positioned where a sulfate is
␮
1
M), DPM-DC (800 nM); 8ꢀpp (13 ␮M), DPM-DC (100 nM); and bound by four highly conserved residues in the crystal struc-
0ꢀpp (11 ␮M), DPM-DC (6.0 ␮M). The reaction mixture con- ture; two of the four residues coordinating this sulfate are con-
ϩ
tained: ATP (2.0 mM, 30ꢀK ), MgCl (3.0 mM), Hepes/K (50 served in S. pneumoniae DPM-DC (Lys-22 and Gly-140), the
m
2
mM, pH 8.0), KCl (50 mM), PK (2.0 units/ml), LDH (4.0 units/ third is conservatively replaced (Lys-74 for Arg-77), and Arg-
ml), PEP (2.0 mM), 2-ME (10 mM), and NADH (0.2 mM, ⌬⑀ 186 replaces the fourth residue, Thr-200. In the T. brucei struc-
ϭ
39
3
Ϫ1
Ϫ1
6
.22 mM cm ); T ϭ 25 Ϯ 2 °C. At the end of each reaction, ture, the helix containing Arg-77 is positioned closer to the
additional equivalents of the analogue and NADH were added sulfate than the corresponding helix in S. pneumoniae, such
to the cuvette, and a second progress curve was obtained; upon that the corresponding Lys-74 is 9 Å from the sulfate. It is there-
comparison to the first curve, it was found to be identical, indi- fore possible that Arg-186 functionally replaces Lys-74. With
cating the absence of product inhibition at the concentrations these considerations, the terminal phosphate of Mevꢀpp was
of product generated by the reaction.
placed within hydrogen bond distance (2.5–4.0 Å) of Lys-22,
Progress Curve Analysis—Values of k and K for each Gly-140, and Arg-186 in the S. pneumoniae model, guided by
cat
m
enzyme with its substrates were determined by statistically fit- the structural alignment. The C -carboxylate, which in the T.
1
JULY 2, 2010•VOLUME 285•NUMBER 27
JOURNAL OF BIOLOGICAL CHEMISTRY 20657

S. pneumoniae Mevalonate Pathway Enzymes
ϩ
brucei model is coordinated by Arg-149, was positioned within Hepes/K (50 mM, pH 8.0), KCl (50 mM), PK (6.0 units/ml),
4
Å of the corresponding Arg-144 and the conserved Ser-141 LDH (12 units/ml), PEP (5.0 mM), 2-ME (10 mM), NADH (10
side chain in the S. pneumoniae model. This Ser residue is con- ␮M), and 6ꢀpp or 9ꢀpp (0, 25, 50 ␮M); T ϭ 25 Ϯ 2 °C. The
served in GHMP kinases that bind Mev, Mevꢀp, and Mevꢀpp and reaction was initiated by addition of Mev; initial velocities were
forms a hydrogen bond with the C -carboxylate in all three measured during consumption of the first 5–7% of the concen-
1
ligand-bound crystal structures (PDB: 2HFU, 2OI2, and tration-limiting substrate (Mev). Under these conditions, prod-
3
GON). The C -hydroxyl group, which is the nucleophile that uct inhibition by Mevꢀp was not observed. The initial velocities,
3
attacks the ATP ␥-phosphate, is positioned in the T. brucei substrate concentrations, and inhibitor concentrations were fit
model near Asp-293, a highly conserved residue in GHMP to a noncompetitive inhibition model (Equation 6).
kinases that assists phosphoryl transfer by deprotonating the
Vmax͓S͔
C -hydroxyl (18). In the S. pneumoniae model, the C -hy-
3
3
V ϭ
(Eq. 6)
droxyl was positioned within 3.5 Å of Asp-276 and points
toward the conserved ATP-binding pocket. To make these
contacts, the C –C bond angle of Mevꢀpp was rotated ϳ120°
I
I
Km
ͩ
1 ϩ
ͪ
ϩ ͓S͔
ͩ
1 ϩ
ͪ
Kis
Kii
2
3
relative to its conformation in the MK crystal structure. This
Diphosphomevalonate Decarboxylase Electrophilic Inactiva-
change puts the C -carboxylate antiperiplanar to the C -hy- tion—Inactivation of S. pneumoniae DPM-DC by putative Xꢀpp
1
3
droxyl that departs during decarboxylation, making the opti- electrophiles (X ϭ 6-10) was tested as a function of turnover.
mal geometry for a concerted elimination reaction, one of Reactions were initiated by the addition of the diphosphory-
the possible mechanisms for this enzyme. Positioned in this lated analogues and allowed to proceed until Ͼ1000 enzyme-
way, the C -methyl group of Mevꢀpp points directly into a equivalents of product had formed. Inactivation was measured
3
large, water-filled cavity composed of Ala-15, Lys-18, Tyr- as a percent reduction in velocity after 1000 turnovers at satu-
1
2
9, Trp-20, Ser-185, Met-189, Met-236, Tyr-249, and Asp- rating substrate. The concentrations of Xꢀpp and DPM-DC in
76. This pocket is highly conserved in each of the seven each assay were: 6ꢀpp (400 ␮M, 133ꢀK ), DPM-DC (10 nM); 7ꢀpp
m
DPM-DC crystal structures available in the PDB.
(350 ␮M, 200ꢀK ), DPM-DC (10 nM); 8ꢀpp (874 ␮M, 156ꢀK ),
m
m
Allosteric Inhibition of Mevalonate Kinase by Mevꢀpp DPM-DC (200 nM); 9ꢀpp (400 ␮M, 200ꢀK ), DPM-DC (100 nM);
m
Analogues—The IC values for the diphosphorylated ana- and 10ꢀpp (400 ␮M, 26.6ꢀK ), DPM-DC (200 nM). The reaction
5
0
m
logues were measured by titration. The reaction mixture mixture contained: ATP (4.0 mM, 54ꢀK ), MgCl (5.0 mM),
m
2
ϩ
included: MK (10 nM), Mev (135 ␮M, 5ꢀK ), ATP (5.0 mM, Hepes/K (50 mM, pH 8.0), KCl (50 mM), PK (3.0 units/ml),
m
ϩ
6
ꢀK ), MgCl (6.0 mM), Hepes/K (50 mM, pH 8.0), KCl (50 LDH (6.0 units/ml), PEP (2.0 mM), 2-ME (10 mM), and NADH
m
2
Ϫ1 Ϫ1
mM), PK (6.0 units/ml), LDH (12 units/ml), PEP (5.0 mM), (200 ␮M, ⌬⑀ ϭ 6.22 mM cm ); T ϭ 25 Ϯ 2 °C. The activity of
2
-ME (10 mM), and NADH (200 ␮M, ⌬⑀ ϭ 6.22 DPM-DC over the course of 1000 turnovers in the presence of
3
39
Ϫ1
Ϫ1
mM cm ); T ϭ 25 Ϯ 2 °C. If no inhibition was observed at Mevꢀpp was used as a negative control for inactivation.
2
50 ␮M, the IC of that compound was not determined.
Monitoring Fate of the Cyclopropyl Ring during DPM-DC
5
0
1
Initial velocities (v) were measured during consumption of Turnover— H NMR was used to monitor the 9ꢀpp cyclopropyl
the first 8–10% of limiting substrate at inhibitor concentra- ring during DPM-DC turnover. The reaction was initiated by
tions ([I]) that were increased until 20–50% inhibition was the addition of 9ꢀpp (3.0 mM) and also contained: DPM-DC (10
ϩ
obtained. The analogue IC values were determined by fit- ␮M), ATP (3.0 mM, 40ꢀK ), MgCl (4.0 mM), Hepes/K (50 mM,
5
0
m
2
ting the v versus [I] data points to Equation 5, where unin- pH 8.0), KCl (50 mM), PK (6.0 units/ml), LDH (12 units/ml),
hibited velocity is given by v .
PEP (5.0 mM), 2-ME (10 mM), NADH (4.0 mM), D O (50 ␮l), and
o
2
3
-trimethylsilyl propionate (3.0 ␮l) as an internal marker. The
1
vo
ϩ ͓I͔/IC50
H NMR spectrum of the reaction mixture was taken at t and
0
v ϭ
(Eq. 5)
1
every 7 min until Ͼ95% conversion of product was reached.
1
13
H- C HSQC and HMBC spectra were taken on the final prod-
To determine the inhibition mechanism and kinetic con- uct to identify carbon functional groups and assign the chemi-
stants for 6ꢀpp and 9ꢀpp inhibition, initial rates were mea- cal shifts of cyclopropyl protons.
sured for a matrix of conditions comprising three inhibitor
All NMR experiments were performed at 25 °C on a Bruker
concentrations and three or four fixed-variable substrate DRX 600-MHz spectrometer equipped with a 5-mm inverse triple
concentrations with the other substrate held constant. resonance probe. One-dimensional proton spectra were collected
When Mev was varied, the assay contained MK (2.5 nM), Mev with64scansof64,000pointsover20ppmandatotalrecycledelay
(
0.5 to 3.7ꢀK ), ATP (3.0 mM, 3.6ꢀK ), MgCl (4.0 mM), of 6 s. The residual water in the spectrum was removed using pre-
m
m
2
ϩ
Hepes/K (50 mM, pH 8.0), KCl (50 mM), PK (3.0 units/ml), saturation of the HOD signal. Spectra were processed with an
LDH (6.0 units/ml), PEP (5.0 mM), 2-ME (10 mM), NADH (10 exponential line broadening of 1 Hz, and the proton chemical
␮
M), 6ꢀpp (0, 15, 40 ␮M), or 9ꢀpp (0, 20, 60 ␮M); T ϭ 25 Ϯ 2 °C. shifts were referenced to internal 3-trimethylsilyl propionate.
1 13
The reactions were initiated with Mev, and after each mea- Two-dimensional H- C HSQC spectra were typically collected
1
13
surement, during which 5–7% of the substrate was con- with 1,000 and 256 complex points in t2 ( H) and t1 ( C), respec-
sumed, Mev was successively added to the cuvette. When tively, with 64 scans per t1 point and a recycle delay of 1.3 s. The
ATP was varied, the assay contained MK (2.5 nM), Mev (135, HSQC experiments used a proton sweep width of 12 ppm and a
13
1
13
5
ꢀK ), ATP (1 to 5ꢀK ), MgCl ([nucleotide] plus 1 mM),
C sweep width of 200 ppm with the H and C carriers set to 4.7
m
m
2
2
0658 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 27•JULY 2, 2010

S. pneumoniae Mevalonate Pathway Enzymes
TABLE 1
Initial rate parameters of S. pneumoniae Mev pathway enzymes with Mev analogues
a
b
c
d
Parentheses indicate Ϯ S.D. as a percentage of the parameter value. Denominator indicates parameter value relative to native acceptor substrate.
Product not detected (see “Experimental Procedures”).
Substrate was not tested.
Poor substrate; initial rate parameters could not determined (see “Experimental Procedures”).
e
X, Xꢀp, and Xꢀpp refer to the non-, mono-, and diphosphorylated mevalonate analogues, respectively.
1
13
and 75 ppm, respectively. Two-dimensional H- C HMBC spec- family kinases (MK, PMK, and DPM-DC), which convert Mev
traweretypicallycollectedwith1,000and256complexpointsint2 to isopentenyl diphosphate, through the metabolic intermedi-
1
13
(
H) and t1 ( C), respectively, with 192 scans per t1 point and a ates Mevꢀp and Mevꢀpp. Because our goal was to use Mev ana-
recycle delay of 1.3 s. The HMBC experiments used a proton logues as prodrugs to generate Mevꢀpp analogues in the cell,
13
sweep width of 12 ppm and a C sweep width of 225 ppm with the knowledge of the substrate selectivities of these enzymes is
1
13
H and C carriers set to 4.7 and 100 ppm, respectively.
helpful in designing potential inhibitors. Toward this end, race-
mic mixtures of ten Mev analogues (1-10, Table 1) were syn-
thesized chemically (9), each with a different substituent
RESULTS AND DISCUSSION
Synthesis of Mev, Mevꢀp, and Mevꢀpp Analogues—The initial replacing the C -methyl of mevalonate. Analogues 1, 3, 6, 7,
3
steps of isoprenoid biosynthesis are catalyzed by three GHMP and 9 (i.e. those with small planar substituents) were enzymat-
JULY 2, 2010•VOLUME 285•NUMBER 27
JOURNAL OF BIOLOGICAL CHEMISTRY 20659

S. pneumoniae Mevalonate Pathway Enzymes
ically phosphorylated to the mono- (Xꢀp) and diphosphorylated reduced k /K values for 4ꢀpp and 5ꢀpp. The substrate selec-
cat
m
(
(
Xꢀpp) forms and purified by anion-exchange chromatography tivity trend in DPM-DC is fundamentally different from that of
see “Experimental Procedures”). Because MK and PMK only MK and PMK, suggesting that substrate recognition is altered in
act on the (R)-isomer (20), the mono- and diphosphorylated this family member. This is perhaps not surprising, given that the
products are stereochemically pure. Pure (R)-Mev analogues Mev moiety is being phosphorylated on a different hydroxyl group
were obtained by treating the (R)-Mevꢀpp analogues (synthe- in DPM-DC, requiring that the geometry of the binding pocket
sized enzymatically) with alkaline phosphatase and purifying as relative to ATP be substantially different from the other enzymes.
above. The monophosphorylated forms of five of the analogues
2, 4, 5, 8, and 10, indicated by a dot (●) in Table 1) could not be
Structural Determinants of Substrate Selectivity—To ascer-
tain whether active-site structural factors might explain the
substrate selectivity of the Mev pathway enzymes, the active
sites of all three enzymes were compared in the vicinity of the
(
produced enzymatically (9), and were not pursued further.
However, racemic mixtures of the diphosphorylated forms of
these analogues were synthesized chemically starting from the
corresponding lactones and used to probe the structural con-
straints of the allosteric pocket of MK and the active site of
DPM-DC (for a full description see supplemental material).
Thus, of the 30 possible analogues (i.e. the non-, mono-, and
diphosphorlyated forms of each of the 10 analogue backbones),
C -methyl group of Mev, Mevꢀp, and Mevꢀpp. In the case of MK
3
and PMK, co-crystal structures of the S. pneumoniae enzymes
with bound acceptor ligands were available (PDB: 2OI2 and
3
GON) (8, 22). There are no ligand-bound structures of DPM-
DC; however, Byres et al. have positioned Mevꢀpp in the T.
brucei DPM-DC. Byres et al. used a number of criteria (18),
2
5 were synthesized, purified, and tested as substrates and allo-
including positioning the C -hydroxyl to attack the ␥-phos-
3
steric inhibitors of the Mev pathway enzymes.
phate of ATP via interactions with conserved resides known to
activate chemistry in GHMP kinases, the presence of an
ordered sulfate interacting with conserved active-site residues
Mevalonate Analogues as Substrates of GHMP Kinases—The
ability of the Mev pathway GHMP kinases to accept alternative
substrates was probed using the 25 Mev analogues listed in
Table 1. Kinetic parameters for each enzyme-substrate pair
were determined by analysis of complete reaction progress
curves. Reactions were monitored using absorbance or fluores-
cence by stoichiometrically coupling (1:1) the production of
ADP to the reduction of NADH, employing the well established
pyruvate kinase/lactate dehydrogenase (PK/LDH) coupled
assay system (12). The activity of PK regenerates the nucleotide,
so the steady-state concentration of ATP is essentially fixed
throughout the reaction. For MK and PMK, phosphorylated
products were removed by the addition of downstream Mev
pathway enzymes, obviating possible product inhibition. For
DPM-DC, progress curves generated in the presence and
absence of decarboxylated products had identical curvatures,
indicating an absence of product inhibition under assay condi-
tions (data not shown). Therefore, product versus time curves
were fit directly to the integrated Michaelis-Menten equation
(
this sulfate was presumed to occupy the binding pocket for the
terminal phosphate of Mevꢀpp) and an Arg that is well posi-
tioned in all DPM-DC structures to interact with the C -car-
1
boxlyate of Mevꢀpp. In addition, we note that, in the three
GHMP kinase structures that contain bound Mev, Mevꢀp, or
Mevꢀpp, the C -carboxylate interacts with a conserved serine.
1
This same Ser is present in each of the seven DPM-DC struc-
tures, and positioning Mevꢀpp according to the Byres et al.
rationale places the carboxylate within contact distance of that
Ser. These conserved anchor points were used to position
Mevꢀpp in our S. pneumoniae model (see Fig. 3C), which was
generated from the crystal structure of S. pyogenes DPM-DC
(
PDB: 2GS8) using Swiss PDB Viewer (17). The sequence of the
S. pyogenes DPM-DC is 70% identical to that of the S. pneu-
moniae enzyme.
A comparison of these models highlights differences in the
active site near the C -methyl group across these enzymes (Fig. 3).
3
(
Equation 1 (21)) to extract k and K (Table 1).
cat
m
The narrow selectivity seen in MK seems likely due to the fact that
Substrate selectivity varied widely across the three GHMP
the C -methyl points into a shallow depression anchored by the
kinases. MK and PMK showed similar trends in catalytic effi-
ciency, accepting small, planar substituents (6) and excluding
larger, branched substituents (2, 4, 5, 8, and 10). However,
PMK generally exhibited lower K and higher k /K values
3
side chain of His-20, a highly conserved MK residue (Fig. 3A) that
appears to be fixed in its orientation by a hydrogen-bonded net-
work that includes the main-chain carbonyls of Gly-256 and His-
m
cat
m
2
0, theamideofVal-23, andacrystallographicwater(Fig. 3A). This
than MK for its substrates (see Table 1), indicating a relaxation
of substrate selectivity in the second step of the pathway.
DPM-DC was the least discriminating of the three enzymes,
accepting all ten analogs as substrates. DPM-DC kinetic param-
eters for 1ꢀpp, 6ꢀpp, and 7ꢀpp were similar to those for Mevꢀpp,
indicating excellent tolerance of a variety of small substituents.
configuration places the C -methyl in van der Waals contact with
3
residues lining the C -binding pocket and leaves little room to
3
admit larger R-groups.
Similar to the interactions seen in the MKꢀMev structure, the
3
C -methyl group of Mevꢀp resides in a shallow, “tight” binding
For larger substituents, branching played an important role in pocket in PMK. In this case, the C -methyl is directed toward a
3
determining substrate selectivity, with unbranched com- tyrosine side chain (Tyr-16) that occupies a position analogous
pounds favored over branched ones. Branched analogs (2ꢀpp, to that of His-20 in MK (Fig. 3B). However, instead of a hydro-
4
ꢀpp, and 10ꢀpp) showed greater than 14-fold decreased affinity gen-bonded network, Tyr-16 is embedded in a hydrophobic
compared with the native substrate, whereas unbranched and pocket composed of Glu-15, Met-216, Val-217, Ile-220, Ile-224,
cyclic analogues (3ꢀpp and 9ꢀpp, respectively) had K values Leu-265, Ile-269, and Ala-293. Although these structures offer
m
similar to that of Mevꢀpp. The binding pocket is apparently little definitive evidence to explain the somewhat relaxed selec-
limited to three-carbon substituents, shown by the strongly tivity of PMK over MK, it is interesting to consider that a hydro-
2
0660 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 27•JULY 2, 2010

S. pneumoniae Mevalonate Pathway Enzymes
FIGURE 3. Structural determinants of substrate selectivity in the mevalonate pathway. Models of the Mev pathway enzymes are shown with their
respective acceptor substrates bound to the active site. Surfaces (light blue) represent the van der Waals contact surface of the protein model. Substrates Mev,
Mevꢀp, andMevꢀppareshowninstickrepresentationandcoloredbyatomtype:Cincyan, Hinwhite, Oinred, andPinorange. TheC -methylgroupisshownwith
3
hydrogens attached. A, S. pneumoniae MK (magenta, PDB: 2OI2) bound to Mev. The Mev C -methyl group points toward His-20, which forms a hydrogen-
3
bonded network (green dashes) with a water molecule (green sphere), Gly-256, and Val-23. B, S. pneumoniae PMK (green, PDB: 3GON) with Mevꢀp. The Mevꢀp
C3-methyl group points toward a hydrophobic network capped by Tyr-19. C, homology model of S. pneumoniae DPM-DC based on the S. pyogenes DPM-DC
(
white, PDB: 2GS8) with Mevꢀpp manually positioned (see “Experimental Procedures”). The Mevꢀpp C -methyl points into a large water-filled cavity composed
3
of nine conserved residues. Images were generated by using PyMOL (19).
phobic pocket may have greater flexibility that one linked by TABLE 2
hydrogen bonds (23, 24).
Steady-state inhibition of S. pneumoniae MK
In contrast to MK and PMK, our model of the DPM-
Inhibitor
IC50
␮M
DCꢀMevꢀpp complex directs the C -methyl group into a deep,
3
Mevꢀpp
0.98 (0.01)
250 (1)
narrow hydrophilic cavity that contains four crystallographic
water molecules in the apo structure (Fig. 3C). Inspection of the
seven DPM-DC homologues in the PDB revealed that the struc-
1
2
ꢀpp
ꢀpp
a
–
3ꢀpp
900 (1)
a
4
ꢀpp
–
ture of the C -cavity and the residues that define it are remark-
a
3
5ꢀpp
6ꢀpp
–
ably well conserved across prokaryotes and eukaryotes. The
volume of the cavity is capable of accommodating two-carbon
R-groups without steric strain, three-carbon R-groups will
likely result in a “snug” fit, whereas larger substituents are unlikely
50 (1)
200 (1)
240 (1)
44 (1)
7
8
ꢀpp
ꢀpp
9ꢀpp
a
1
0ꢀpp
–
a
Compound inhibited enzyme turnover by Ͻ1% at a concentration of 250 ␮M.
to access the pocket well. This simple analysis of the C -pocket
3
Parenthetical values indicate standard error.
accessibility predicts the behavior of the enzyme toward the C
3
TABLE 3
analogues well (see Table 1) and supports the validity of the model.
It is interesting to consider that this expanded cavity, which
Initial rate parameters for S. pneumoniae MK in the presence of Xꢀpp
Inhibitor Km-Mev Kis-Meva Kii-Mev
Km-ATP
Kis-ATP Kii-ATP
extends the substrate repertoire of the enzyme at C , may provide
3
␮
M
␮M
␮M
mM
␮M
␮M
the cell with the means to decarboxylate other, as yet unidentified,
b
6
ꢀpp
ꢀpp
25 (1)
23 (3)
76 (2)
58 (3)
75 (3) 0.98 (0.05)
43 (6)
59 (6)
45 (3)
65 (5)
␤
-hydroxy carboxylic acids.
9
55 (5) 0.98 (0.05)
Mevꢀpp Analogues as Allosteric Inhibitors of MK—The ten
a
b
K
and K are the steady-state affinities of the inhibitor for the EꢀS2 and EꢀS1ꢀS2
is
ii
complexes, respectively, where S1 and S2 are the non-variable and variable sub-
diphosphorylated analogues (Table 1) were designed to probe
the structure of the allosteric site of MK. A preliminary study
was carried out in which all ten compounds were screened at
strates, respectively (see “Experimental Procedures”).
Standard errors are enclosed in parentheses.
2
50 ␮M for MK inhibition at fixed concentrations of substrates. initial rate study in which the concentration of one substrate
Compounds that inhibited MK activity by Ͻ1% at this level (Mev or ATP, four values spanning the K values) was varied
m
(
2ꢀpp, 4ꢀpp, 5ꢀpp, and 10ꢀpp) were not considered further. For against the concentration of inhibitor (three values spanning
compounds exhibiting detectable inhibition at 250 ␮M, a titra- the K ) (see supplemental Fig. S1). A noncompetitive reversible
i
tion was carried out to determine the IC (Table 2 and “Exper- inhibition model provided the best fit to each data set (Table 3).
50
imental Procedures”). MK inhibitors fell into three broad Pure noncompetitive inhibition by 6ꢀpp and 9ꢀpp against both
groups based on their IC values relative to that of Mevꢀpp: substrates, as indicated by identical K and K values (within
50
is
ii
ϳ50-fold higher (6ꢀpp and 9ꢀpp), ϳ250-fold higher (1ꢀpp, 7ꢀpp, error), is diagnostic of an allosteric inhibition mechanism (13,
and 8ꢀpp), and ϳ900-fold higher (3ꢀpp). Although we expect 25). These results indicate that Xꢀpp inhibition is due to binding
that this inhibition represents binding of a ligand at the allo- at the allosteric site.
steric site, it is formally possible that the analogue (Xꢀpp), which
The Xꢀpp analogues are relatively weak inhibitors, suggesting
resembles the acceptor (X) bound to the ␤- and ␥-phosphates that the allosteric binding pocket does not tolerate substitution
of ATP, could act as a bisubstrate inhibitor, in which case it is of the Mevꢀpp C -methyl group by larger moieties. The shape of
3
predicted to compete with the substrates. To distinguish the pocket is difficult to infer, because the IC values do not
5
0
between these possibilities, the inhibition mechanism of the correlate with the size or flexibility of the C substituents. For
3
two best inhibitors, 6ꢀpp and 9ꢀpp, was determined in a classic example, among analogues with two-carbon substituents, the
JULY 2, 2010•VOLUME 285•NUMBER 27
JOURNAL OF BIOLOGICAL CHEMISTRY 20661

S. pneumoniae Mevalonate Pathway Enzymes
Ͼ1000 turnovers of the enzyme.
With each analogue, the rates of
reaction before and after 1000 turn-
overs were identical, within experi-
mental error, indicating that Յ4% of
the enzyme was inactivated during
the measurement. Thus, either the
analogues are decarboxylated (like
Mevꢀpp) or the carbocation reacts
with water to form non-decarboxy-
lated products.
To determine the fate of the ana-
logues, we focused on the cyclopro-
pyl derivative (9ꢀpp), which we
expected to inactivate DPM-DC via
opening of the strained three-mem-
bered ring (28). When a tertiary car-
bocation forms adjacent to a cyclo-
propyl ring, it is expected to
1
FIGURE4. NMRdeterminationofthefateofthecyclopropylring. A, partialone-dimensional HNMRspectra rearrange into a homoallyl cation
at 600 MHz obtained at various times during the enzymatic reaction of 9ꢀpp with DPM-DC. A spectrum was
(
see Fig. 2) that is stabilized due to
taken every 7 min after the addition of enzyme. The time stamp of a given reaction time is shown. The multi-
plets at 0.40 ppm/0.35 ppm and at 0.69 ppm/0.50 ppm represent the cyclopropyl methylene protons of the
release of 27 kcal/mol ring strain
startingmaterialandproduct, respectively. Theintegratedareasofthemultipletsarepreservedduringthereaction (29, 30). If the homoallyl cation were
1
13
indicating that all starting material is converted to product. B, expansion of two-dimensional H- C HSQC with
to react with water (rather than the
protein) at the active site, the reac-
tion would produce a ring-opened
primary alcohol that can be readily
1
13
multiplicity editing of the product after enzymatic reaction of 9ꢀpp. The signals at 0.69 ppm ( H)/5.5 ppm ( C) and
at 0.50 ppm ( H)/5.5 ppm ( C) are from the methylene protons of the cyclopropyl ring. These chemical shifts are
characteristic of a cyclopropyl ring and demonstrate that the ring is preserved during the enzymatic reaction.
1
13
vinyl analogue (6ꢀpp) inhibits MK 4- to 5-fold better than either identified by NMR spectrometry due to the large chemical shift
the ethyl (1ꢀpp) or ethynyl (7ꢀpp) analogues, suggesting that differences between cyclopropyl protons and their linear coun-
some subtle combination of size, geometry, and electrophilicity terparts (31). An NMR experiment that monitored the cyclo-
makes the vinyl group preferred. Analogues with three-carbon propyl proton resonances during product formation revealed
substituents appear to inhibit differently on the basis of shape: that the substrate peaks are shifted only slightly downfield
linear 8ꢀpp outperforms the more flexible 3ꢀpp. Surprisingly, (ϳ0.3 ppm) in the product. The sum of the integrated intensi-
the vinyl and cyclopropyl (9ꢀpp) analogues inhibit equally well, ties of the substrate and product resonances remained fixed
despite the addition of a carbon in 9ꢀpp and the exclusion of all throughout the experiment (Fig. 4). An HSQC experiment
other branched substituents. This result suggests that the identified the product resonances (0.5 and 0.68 ppm, Fig. 4A) as
region of the allosteric site that binds the C substituent is those of cyclopropyl ring protons (Fig. 4B). Furthermore, the
3
highly sensitive to the geometry of the group and that favorable cyclopropyl protons in the product showed long range correla-
electronic interactions between the enzyme and the ␲-orbitals tions to carbons at 107.6 ppm and 148.3 ppm (two-dimensional
1
13
in the vinyl group and the cyclopropyl ring may contribute to
binding affinity.
H- C HMBC (32); data not shown), indicating that a double
bond is adjacent to the cyclopropyl ring, which confirmed that
This study revealed that our prodrug strategy for MK inhibi- the product results from decarboxylation. These NMR data rule
tion will be limited to Mev analogues with small C substitu- out a ring-opened product and support the analogue undergo-
3
ents. Fluorinated Mevꢀpp analogues, which are isosteric with ing a decarboxylation that resembles that of the native sub-
Mevꢀpp, are inhibitors of mammalian DPM-DC and should be strate, Mevꢀpp.
excellent inhibitors of S. pneumoniae MK (26, 27).
These results call into question the extent of carbocation
Mevꢀpp Analogues as Probes of DPM-DC Reaction Mecha- formation in the DPM-DC transition state. If a full carbocation
nism—Five analogues (6ꢀpp through 10ꢀpp) were designed to had formed, we expected to observe rearrangement of the
covalently inactivate DPM-DC by forming a highly reactive car- cyclopropyl group. If no nucleophile is in close proximity to the
bocation capable of covalent attachment at the active site (see carbocation (the protein surface or water), we might expect to
the introduction and Fig. 2). These compounds did not detect- see only intact cyclopropyl products; however, the presence of
ably inactivate the enzyme under the initial rate conditions four crystallographic water molecules in the large pocket adja-
used to measure their kinetic constants as substrates of cent to C and the proximity of the Asp-276, Lys-18, Ser-185,
3
DPM-DC (see “Mevalonate Analogues as Substrates of GHMP and Met-189 side chains and the Tyr-19 carbonyl as potential
Kinases”). To assess whether inactivation was too slow to detect nucleophiles argue against this possibility (Fig. 3C). Alterna-
in the initial rate experiments, the DPM-DC reaction condi- tively, carbocation formation may be minimal or absent, in
tions were adjusted (see “Experimental Procedures”) so that the which case elimination of the carboxylate and the phosphate is
concentration of the analogue would remain saturating during concerted rather than dissociative. Abeles’ work on the effects
2
0662 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 27•JULY 2, 2010

S. pneumoniae Mevalonate Pathway Enzymes
of altered electron induction at C with the mammalian enzyme
3. Van Bambeke, F., Reinert, R. R., Appelbaum, P. C., Tulkens, P. M., and
3
Peetermans, W. E. (2007) Drugs 67, 2355–2382
shows clearly that DPM-DC chemistry is sensitive to such
changes and supports the development of a positive charge at
C in the transition state (10). Further, by replacing C with a
4
. Kyaw, M. H., Lynfield, R., Schaffner, W., Craig, A. S., Hadler, J., Reingold,
A., Thomas, A. R., Harrison, L. H., Bennett, N. M., Farley, M. M., Facklam,
R. R., Jorgensen, J. H., Besser, J., Zell, E. R., Schuchat, A., and Whitney,
C. G. (2006) N. Engl. J. Med. 354, 1455–1463
3
3
positively charged amine, he created an analogue that mim-
icked the structure and charge characteristics of a dissociative
transition state. The affinity of this analogue (0.75 ␮M) was only
5. Huang, S. S., Hinrichsen, V. L., Stevenson, A. E., Rifas-Shiman, S. L., Klein-
man, K., Pelton, S. I., Lipsitch, M., Hanage, W. P., Lee, G. M., and Finkel-
stein, J. A. (2009) Pediatrics 124, e1–e11
2
0-fold higher than that of the substrate (33), which, while sup-
6
. Wilding, E. I., Brown, J. R., Bryant, A. P., Chalker, A. F., Holmes, D. J.,
Ingraham, K. A., Iordanescu, S., So, C. Y., Rosenberg, M., and Gwynn,
M. N. (2000) J. Bacteriol. 182, 4319–4327
portive of a dissociative character in the transition state, is per-
haps more consistent with development of partial rather than
complete positive charge at C . Although studies that correlate
3
7. Lange, B. M., Rujan, T., Martin, W., and Croteau, R. (2000) Proc. Natl.
Acad. Sci. U.S.A. 97, 13172–13177
theextentofpositivechargeformationwithdegreeofringopening
in cyclopropyl ring systems do not yet exist, our results, which
demonstrate no detectible ring opening, are consistent with only
slight positive charge formation in the transition state. Using
kinetic isotope effects, it may be possible to assess the extent of
8. Andreassi, J. L., 2nd, Bilder, P. W., Vetting, M. W., Roderick, S. L., and
Leyh, T. S. (2007) Protein Sci. 16, 983–989
9
. Kudoh, T., Park, C. S., Lefurgy, S. T., Sun, M., Michels, T., Leyh, T. S., and
Silverman, R. B. (2010) Bioorg. Med. Chem. 18, 1124–1134
1
1
0. Dhe-Paganon, S., Magrath, J., and Abeles, R. H. (1994) Biochemistry 33,
positive charge development on C at the transition state.
3
1
3355–13362
Targeting the Human DPM-DC—Exclusive of their effects on
1. Pilloff, D., Dabovic, K., Romanowski, M. J., Bonanno, J. B., Doherty, M.,
bacterial enzymes, the Mevꢀpp analogues might also inhibit the
Burley, S. K., and Leyh, T. S. (2003) J. Biol. Chem. 278, 4510–4515
human DPM-DC, because it is not clear whether the corre- 12. Tchen, T. T. (1958) J. Biol. Chem. 233, 1100–1103
1
3. Andreassi, J. L., 2nd, Dabovic, K., and Leyh, T. S. (2004) Biochemistry 43,
sponding human and bacterial enzymes have diverged to the
point where they could be targeted orthogonally, as is the case
for MK. Inhibitors of the human Mev pathway (statins and
bisphosphonates) are used clinically to reduce cholesterol bio-
synthesis, increase bone density, and decrease cell proliferation
1
6461–16466
1
1
1
4. McClure, W. R. (1969) Biochemistry 8, 2782–2786
5. Cleland, W. W. (1967) Adv. Enzymol. Relat. Areas Mol. Biol. 29, 1–32
6. Mandel, J. (1964) The Statistical Analysis of Experimental Data, pp.
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31–159, Dover, New York
in cancer (34). Down-regulation of the human Mev pathway by 17. Guex, N., and Peitsch, M. C. (1997) Electrophoresis 18, 2714–2723
1
1
2
8. Byres, E., Alphey, M. S., Smith, T. K., and Hunter, W. N. (2007) J. Mol. Biol.
statins has recently been linked to disruption of replication by
hepatitis C virus (35, 36) and human immunodeficiency virus
371, 540–553
9. DeLano, W. L. (2002) The PyMOL Molecular Graphics System, DeLano
Scientific LLC, San Carlos, CA
0. Cornforth, R. H., Fletcher, K., Hellig, H., and Popjak, G. (1960) Nature 185,
(
37), enhancement of anticancer drugs (38), and anti-inflam-
matory effects in the lung and airways (39, 40). These findings
suggest that DPM-DC inhibitors may find additional uses in the
9
23–924
treatment of non-infectious diseases, which obviates the need 21. London, J. W., Shaw, L. M., and Garfinkel, D. (1977) Anal. Chem. 49,
for selective DPM-DC inhibition.
1716–1719
2
2
2
2. Andreassi, J. L., 2nd, Vetting, M. W., Bilder, P. W., Roderick, S. L., and
Leyh, T. S. (2009) Biochemistry 48, 6461–6468
Conclusions—Twenty-five novel Mev analogues have been
tested as substrates and inhibitors of three enzymes that com-
prise the Mev pathway in S. pneumoniae. Although the MK
allosteric binding pocket admits certain analogues, it is highly
selective for Mevꢀpp. Substrate selectivity of the enzymes varies
3. Davis, A. M., and Teague, S. J. (1999) Angew. Chem. Int. Ed. Engl. 122,
7
36–749
4. Kawaguchi, S., Nobe, Y., Yasuoka, J., Wakamiya, T., Kusumoto, S., and
Kuramitsu, S. (1997) J Biochem. 122, 55–63
considerably across the pathway, with MK providing the most 25. Cleland, W. W. (1963) Biochim. Biophys. Acta 67, 173–187
26. Qiu, Y., and Li, D. (2006) Biochim. Biophys. Acta 1760, 1080–1087
27. Reardon, J. E., and Abeles, R. H. (1987) Biochemistry 26, 4717–4722
28. Suckling, C. J. (1988) Angew. Chem. Int. Ed. Engl. 27, 537–552
29. Hart, H., and Sandri, J. M. (1959) J. Am Chem. Soc. 81, 320–326
30. Liebman, J. F., and Greenberg, A. (1976) Chem. Rev. 76, 311–365
31. Gunther, H. (1997) NMR Spectroscopy: Basic Principles, Concepts, and
Applications in Chemistry, John Wiley & Sons, New York
stringent selection. The active-site structures of the Mev path-
way GHMP kinases provide a rationale for the substrate selec-
tivity of these enzymes toward substitution at C . The C
3
3
R-group pocket in DPM-DC is considerably larger than that of
MK or PMK, and the volume of this cavity correlates well with
selectivity toward R-group substitution. The high conservation
of this pocket indicates that it has been evolutionarily maintained, 32. Bax, A., and Summers, M. F. (1986) J. Am Chem. Soc. 108, 2093–2094
3
3
3
3. Wolfenden, R. (1999) Bioorg. Med. Chem. 7, 647–652
and that the ability to decarboxylate substrates that vary at the C
3
4. Buhaescu, I., and Izzedine, H. (2007) Clin. Biochem. 40, 575–584
5. Lyn, R. K., Kennedy, D. C., Sagan, S. M., Blais, D. R., Rouleau, Y., Pegoraro,
A. F., Xie, X. S., Stolow, A., and Pezacki, J. P. (2009) Virology 394, 130–142
6. Ye, J., Wang, C., Sumpter, R., Jr., Brown, M. S., Goldstein, J. L., and Gale,
M., Jr. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 15865–15870
R-group may provide an important metabolic function. Finally,
resultsusinganaloguesdesignedtoactasmechanism-basedinhib-
itors of DPM-DC suggest that ionization of phosphate and decar-
boxylation of the pꢀMevꢀpp intermediate occurs in a concerted
3
fashion with little carbocation development at C .
37. del Real, G., Jim e´ nez-Baranda, S., Mira, E., Lacalle, R. A., Lucas, P., G u¨ mez-
Mout u¨ n, C., Alegret, M., Pe n˜ a, J. M., Rodríguez-Zapata, M., Alvarez-Mon,
M., Martínez, A. C., and Ma n˜ es, S. (2004) J. Exp. Med. 200, 541–547
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JULY 2, 2010•VOLUME 285•NUMBER 27
JOURNAL OF BIOLOGICAL CHEMISTRY 20663

Enzymology:
Probing Ligand-binding Pockets of the
Mevalonate Pathway Enzymes from
Streptococcus pneumoniae
Scott T. Lefurgy, Sofia B. Rodriguez, Chan
Sun Park, Sean Cahill, Richard B. Silverman
and Thomas S. Leyh
J. Biol. Chem. 2010, 285:20654-20663.
doi: 10.1074/jbc.M109.098350 originally published online April 19, 2010
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