From the Department of Microbiology, Colorado State
University, Fort Collins, Colorado 80523 and the
§ Universitá di Pisa, Dipartimento di Scienze
Farmaceutiche, Via Bonanno 6, Pisa 56126, Italy
Received for publication, August 8, 2000, and in revised form, November 28, 2000
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ABSTRACT |
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We have recently shown that open reading frame
Rv1086 of the Mycobacterium tuberculosis H37Rv genome
sequence encodes a unique isoprenyl diphosphate synthase. The product
of this enzyme, Isoprenyl diphosphate synthases catalyze the condensation of an
allylic diphosphate with isopentenyl diphosphate
(IPP,1 C5) via an
electrophilic alkylation reaction to produce longer allylic
diphosphates (1, 2). Chain elongation continues until a
physiologically appropriate chain length is reached, at which time the
molecule may undergo further modifications (dephosphorylation, cyclization, or head-to-head condensation reactions). Polyprenyl phosphate (Pol-P) is formed by dephosphorylation of an allylic prenyl
diphosphate chain. The predominant form of prokaryotic Pol-P is
Pol-P is central to prokaryotic cell wall synthesis as a sugar carrier,
and it has been reported that the levels of Pol-P may be rate-limiting
for in vivo cell wall synthesis (10-13). Our laboratory has
shown that Pol-P is instrumental in the synthesis of each component of
the covalently linked peptidoglycan-arabinogalactan-mycolic acid cell
wall core of mycobacteria, and other noncovalently associated macromolecules such as lipomannan and lipoarabinomannan (5, 14, 15).
The importance of Pol-P is also demonstrated in vivo by the
fact that M. tuberculosis (16) (and other
Mycobacterium sp.)3 are sensitive to the
antibiotic bacitracin, which specifically binds isoprenyl diphosphate
(17) intermediates in Pol-P synthesis, inhibiting both chain elongation
and dephosphorylation reactions.
Evolutionarily, there appear to be two independent families of
isoprenyl diphosphate synthases, based on the type of stereochemistry (E or Z) introduced at the products' new double
bond. E-Isoprenyl diphosphate synthases are capable of
catalyzing the chain elongation of a range of substrates, the smallest
one being dimethylallyl diphosphate (DMAPP, C5) with IPP to
form Thus far, only seven protein sequences have been biochemically
correlated with Z-isoprenyl diphosphate synthase activity, including undecaprenyl diphosphate synthases from Micrococcus luteus, Escherichia coli, Hemophilus
influenzae, Streptococcus pneumoniae, the dolichol
synthase from Saccharomyces cerevisiae, and the decaprenyl
diphosphate and farnesyl diphosphate synthases from M. tuberculosis. Prior to the identification of the
Z-farnesyl diphosphate (Z-FPP) synthase from
M. tuberculosis (9), all known Z-isoprenyl
diphosphate synthases utilized
The crystal structure of the short-chain E-isoprenyl
diphosphate synthase (avian FPP synthase) has been determined (18), and
mutagenesis studies have been performed (19-21) providing a solid
understanding of how the active site determines the chain length of the
product (22, 23). However, little is known about Z-isoprenyl
diphosphate synthases. We have purified and enzymatically characterized
the short-chain Z-isoprenyl diphosphate synthase from
M. tuberculosis, which catalyzes the first committed step in
the synthesis of decaprenyl diphosphate, a molecule whose role in cell
wall synthesis is likely essential.
Synthesis
All oxygen- and water-sensitive reactions (Scheme
1) were performed under dry argon
atmosphere. 1H NMR spectra of all compounds were
obtained with a Varian Gemini 200 operating at 200 MHz in approximately
2% solution of CDCl3 or D2O, using
Me4Si or
Me3Si(CH2)3SO3Na as the
internal standard. Column chromatographies were performed using 230- to
400-mesh silica gel (Merck) or reverse phase silica gel (Macherey-Nagel Polygosil 60-4063 C18). Mass spectra were recorded on a VG
70-25S mass spectrometer or an HP-5988 A spectrometer. Reagents and
solvents were purchased from Aldrich or Fluka.
,E,Z-farnesyl diphosphate, is an intermediate for the synthesis of decaprenyl phosphate, which has a central role in the biosynthesis of most features of the mycobacterial cell wall, including peptidoglycan, arabinan, linker unit galactan, and lipoarabinomannan. We have now
purified Z-farnesyl diphosphate synthase to near
homogeneity using a novel mycobacterial expression system.
Z-Farnesyl diphosphate synthase catalyzed the addition of
isopentenyl diphosphate to
,E-geranyl
diphosphate or
,Z-neryl diphosphate yielding
,E,Z-farnesyl diphosphate and
,Z,Z-farnesyl diphosphate,
respectively. The enzyme has an absolute requirement for a divalent
cation, an optimal pH range of 7-8, and Km values
of 124 µM for isopentenyl diphosphate, 38 µM for geranyl diphosphate, and 16 µM for
neryl diphosphate. Inhibitors of the Z-farnesyl diphosphate
synthase were designed and chemically synthesized as stable analogs of
,E-geranyl diphosphate in which the labile
diphosphate moiety was replaced with stable moieties. Studies with
these compounds revealed that the active site of Z-farnesyl
diphosphate synthase differs substantially from E-farnesyl
diphosphate synthase from pig brain (Sus
scrofa).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,diE,polyZ-undecaprenyl
phosphate2 (C55);
however, there are documented exceptions in Paracoccus denitrificans (3) and in Mycobacterium sp. (4-8).
M. smegmatis contains heptaprenyl diphosphate (8)
(C35, four saturated, three Z double bonds) and
decaprenyl diphosphate (5) (C50,
, one
E, and eight Z double bonds), whereas M. tuberculosis contains only decaprenyl phosphate (6). Although the
stereochemistry of decaprenyl phosphate from M. tuberculosis
has not been determined, our enzymatic studies suggest that it has
similar stereochemistry to decaprenyl phosphate from M. smegmatis (9).
,E-geranyl diphosphate (
,E-GPP, C10). Other short-chain
isoprenyl diphosphates
(
,E,E-farnesyl diphosphate
(
,E,E-FPP, C15) and
,E,E,E-geranylgeranyl
diphosphate (
,E,E,E-GGPP,
C20)) are generated by a similar mechanism with additional
molecules of IPP. Medium-chain E-isoprenyl diphosphate synthases use the short-chain products as allylic substrates to produce
compounds that are C30 to C50 in length.
Medium-chain E-isoprenyl diphosphate synthases are
homologous to the short-chain E-isoprenyl diphosphate
synthases, because both types contain two signature aspartate motifs
(DD(XX)1-2D).
,E,E-FPP or
,E,E,E-GGPP as the
allylic substrate, added multiple units of IPP, and released long-chain
(C45 and greater) isoprenyl diphosphate molecules with
mixed stereochemistry (2).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Scheme 1.
Synthesis of
,E-geranyl diphosphate analogs,
Compounds 1-4. Reagents and conditions: (i)
N-hydroxyphthalimide, diethyl azodicarboxylate,
triphenylphosphine, anhydrous tetrahydrofuran, 18 h, room
temperature; (ii) NH2NH2, ethanol, 18 h,
room temperature; (iii) 1-hydroxybenzotriazole,
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride,
anhydrous tetrahydrofuran, 18 h, room temperature; (iv) a,
bromotrimethylsilane, 2,4,6-collidine, dichloromethane, 18 h, room
temperature; b, KOH 1 N, 3 h, room temperature; (v)
butyl lithium, 1.6 M hexane solution, anhydrous
tetrahydrofuran, 1 h,
78 °C.
(E)-N-(3,7-Dimethyl-2,6-octadienyloxy)phthalimide
(6)--
,E-Geraniol 5 (2.0 g,
13 mmol) was added to a solution of N-hydroxyphthalimide
(2.12 g, 13 mmol), triphenylphosphine (3.42 g, 13 mmol), and diethyl
azodicarboxylate (2.25 ml, 14.3 mmol) in anhydrous tetrahydrofuran (50 ml). After stirring for 18 h at room temperature, the solvent was
evaporated and the residue was purified on silica gel, eluting with
CH2Cl2-hexane (6:4). The appropriate fractions
were combined and evaporated to give the intermediate 6 (3.19 g, 82%) as a white solid. Elemental analysis of compound
6 was consistent with theoretical values to within ±0.4%,
m.p. 89-91 °C (uncorrected); 1H NMR
(CDCl3):
1.57 (s, 3H, CH3), 1.64 (s, 3H,
CH3), 1.71 (s, 3H, CH3), 1.93-2.18 (m, 4H,
2 × CH2), 4.72 (d, 2H, J = 8 Hz,
CH2), 5.01 (br, 1H, CH), 5.53 (t, 1H,
J = 8 Hz, CH), 7.60-7.85 (m, 4H, Ar); Analysis for
C18H21NO3: C, 71.93; H, 7.15; N,
4.59. Found: C, 72.15; H, 7.02; N, 4.68.
(E)-3,7-Dimethyl-2,6-octadienyl-1-oxyamine
(7)--
Hydrazine monohydrate (0.69 ml, 14.22 mmol) was
added to a solution of 6 (2.13 g, 7.11 mmol) in ethanol (200 ml), and the resulting mixture was stirred at room temperature for
18 h. After filtration of the white solid formed, the solution was
evaporated and the resulting crude residue was extracted with diethyl
ether (3 × 100 ml). The diethyl ether was removed by evaporation
to give 7 (1.09 g, 91%) as an oil, which was used for the next reactions without further purification. 1H NMR
(CDCl3): 1.60 (s, 3H, CH3), 1.68 (s, 6H,
2 × CH3), 1.96-2.18 (m, 4H, 2 × CH2), 4.17 (d, 2H, J = 6.4 Hz,
CH2), 5.05 (m, 1H, CH), 5.31 (t, 1H, J = 6.4 Hz, CH); MS m/e 170 (M+H)+.
2-(Diethylphosphono)butyric Acid (9)--
A solution
containing KOH (2.35 g, 42 mmol), absolute ethanol (7 ml), and water (3 ml) was added dropwise to triethyl 2-phosphonobutyrate (9.9 ml, 42 mmol), and the resulting mixture was stirred at room temperature for
24 h. The solvents were removed under reduced pressure and the
solid residue was triturated with diethyl ether (3 × 130 ml),
which was discarded. The residue was dissolved in water (30 ml) and
acidified to pH 1 with HCl 6 M. The solution was then
saturated with solid NaCl and extracted with
CH2Cl2 (3 × 25 ml). The organic phase was
dried and evaporated to give intermediate 9 (8.5 g, 90%) as
an oil. 1H NMR (CDCl3): 1.01 (t, 3H,
J = 7.2 Hz, CH3CH2),
1.32 (t, 6H, J = 7.2 Hz, 2 × CH3CH2), 1.53-2.15 (m, 2H,
CH3CH2), 2.87 (dt, 1H,
J = 7.2, 22 Hz, CH), 3.92-4.38 (m, 4H, 2 × CH3CH2); MS
m/e 225 (M+H)+.
Diethyl
(E)-[2-Oxo-2-[[(3,7-dimethyl-2,6-octadienyl)oxy]amino]ethyl]Phosphonate
(10) and Diethyl
(E)-1-[[(3,7-Methyl-2,6-octadienyl)oxy]aminocarbonyl]propyl
Phosphonate (11)A solution of compound 7(0.340 g, 2.03 mmol), 8 (24), or 9 (2.23 mmol),
and 1-hydroxybenzotriazole (0.410 g, 3.04 mmol) in anhydrous
tetrahydrofuran (17 ml) was treated with
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.470 g,
2.44 mmol). The mixture was stirred at room temperature for 8 h,
and the solvent was evaporated under reduced pressure. The residue was
purified by silica gel column chromatography, eluting with ethyl
acetate-hexane in a ratio of 2:1 in the case of 10 and in a
ratio of 1:1 in the case of 11. The appropriate fractions
were combined, evaporated, and pump-dried to give the appropriate
intermediate 10 or 11 as an oil. 10 (0.493 g, 70%) 1H NMR (CDCl3):
1.32 (t,
6H, J = 7.2 Hz, 2 × CH3CH2), 1.59 (s, 3H,
CH3), 1.70 (s, 6H, 2 × CH3), 1.93--
2.18
(m, 4H, 2 × CH2), 2.82 (d, 2H, J = 21 Hz, CH2P), 4.08 (q, 4H, J = 7.2 Hz, 2 × CH3CH2), 4.42 (d, 2H,
J = 7.2 Hz, CH2), 5.04 (m, 1H, CH), 5.37 (t, 1H, J = 7.2 Hz, CH); MS (FAB+)
m/e 348 (M+H)+. 11 (0.530 g, 69%) 1H NMR (CDCl3):
0.99 (t, 3H,
J = 7.2 Hz, CH3CH2),
1.28 (t, 3H, J = 7.2 Hz,
CH3CH2), 1.30 (t, 3H,
J = 7.2 Hz, CH3CH2),
1.59 (s, 3H, CH3), 1.69 (s, 6H, CH3),
1.85-2.21 (m, 6H, 2 × CH2 + CH3CH2), 2.55 (dt, 1H,
J = 7.2, 22 Hz, CH), 3.91-4.27 (m, 4H, 2 × CH3CH2), 4.41 (d, 2H,
J = 7.2 Hz, CH2), 5.07 (br, 1H, CH), 5.38 (t, 1H, J = 7.2 Hz, CH); MS (FAB+)
m/e 376 (M+H)+.
Dipotassium Salt of
(E)-[2-Oxo-2-[[(3,7-methyl-2,6-octadienyl)oxy[amino]ethyl]Phosphonic
Acid (1)--
Bromotrimethylsilane (0.790 ml, 6 mmol) was
added to a stirred solution of compound 10 (0.416 g, 1.2 mmol) and 2,4,6-collidine (0.316 ml, 2.4 mmol) in anhydrous
CH2Cl2 (12 ml); the resulting mixture was
stirred at room temperature for 18 h. After evaporation of the
solution, the residue was treated with an aqueous solution of KOH (1 N, 9 ml) and then stirred at room temperature for 3 h.
The solution was evaporated, and the resulting crude residue was
purified by column chromatography on reverse phase silica gel, by
eluting with methanol-water (1:4) and collecting 2-ml fractions. The
appropriate fractions were combined, evaporated, lyophilized, and
pump-dried to give 1 (0.313 g, 71%) as a very hygroscopic
white lyophilate: 1H NMR (D2O): 1.58 (s,
3H, CH3), 1.64 (s, 3H, CH3), 1.68 (s, 3H,
CH3), 2.05-2.15 (m, 4H, 2 × CH2), 2.40 (d, 2H, J = 19 Hz, CH2P), 4.38 (d, 2H,
J = 7.2 Hz, CH2), 5.16 (br, 1H, CH), 5.39 (t, 1H, J = 7.2 Hz, CH); MS (FAB+)
m/e 368 (M+H)+.
Dipotassium Salt of
(E)-1-[[(3,7-Methyl-2,6-octadienyl)oxy]aminocarbonyl]propyl
Phosphonic Acid (2)--
Compound 2 was
prepared following the experimental procedure reported for 1. 2 (0.322 g, 68%) 1H NMR (D2O): 0.87 (t, 3H, J = 7.2 Hz,
CH3CH2), 1.62 (s, 3H, CH3), 1.69 (s, 3H, CH3), 1.71 (s, 3H,
CH3), 2.01-2.29 (m, 6H, 2 × CH2 + CH3CH2), 2.41-2.62 (m, 1H, CH),
4.40 (d, 2H, J = 7.2 Hz, CH2), 5.18 (br,
3H, 3 × CH), 5.40 (t, 1H, J = 7.2 Hz, CH); MS
(FAB+) m/e 396 (M+H)+.
Diethyl
(E)-1-[(3,7-Methyl-2,6-octadienyl)aminocarbonyl]propyl Phosphonate
(13)--
A solution of ,E-geranylamine
12 (0.370 g, 2.42 mmol), was treated with 9 (0.600 g, 2.66 mmol) to produce 11 following the
experimental procedure reported for 10. The resulting crude
residue was purified on silica gel, eluting with ethyl acetate-hexane
(2:3). The appropriate fractions were combined, evaporated, and
pump-dried to give intermediate 13 (0.770 g, 88%) as an
oil; 1H NMR (CDCl3):
1.00 (t, 3H,
J = 7.2 Hz, CH3CH2),
1.30 (t, 6H, J = 7.2 Hz, 2 × CH3CH2), 1.59 (s, 3H,
CH3), 1.66 (s, 3H, CH3), 1.69 (s, 3H,
CH3), 1.85-2.25 (m, 6H, 2 × CH2 + CH3CH2), 2.72 (dt, 1H,
J = 7.2, 22 Hz, CH), 3.81 (d, 2H, J = 7.2 Hz, CH2), 4.05 (q, 4H, J = 7.2 Hz,
2 × CH3CH2), 5.07 (br, 3H,
3 × CH), 5.18 (t, 1H, J = 7.2 Hz, CH); MS
(FAB+) m/e 360 (M+H)+.
Dipotassium Salt of
(E)-1-[(3,7-Methyl-2,6-octadienyl)aminocarbonyl]propyl Phosphonic
Acid (3)--
Compound 3 was prepared following
the experimental procedure reported for 1. In this case, the
resulting crude residue was purified by column chromatography on
reverse phase silica gel, eluting with methanol-water in a 2:3 ratio.
3 (0.409 g, 90%) 1H NMR (D2O): 0.87 (t, 3H, J = 7.2 Hz,
CH3CH2), 1.63 (s, 3H, CH3), 1.67 (s, 3H, CH3), 1.70 (s, 3H,
CH3), 1.99-2.18 (m, 6H, 2 × CH2 + CH3CH2), 2.22-2.49 (m, 1H, CH),
3.41 (d, 2H, J = 7.2 Hz, CH2), 5.12 (br, H,
CH), 5.23 (t, 1H, J = 7.2 Hz, CH); MS
(FAB+) m/e 380 (M+H)+.
Diethyl
[(E)-1-Ethyl-1-[(4,8-dimethyl-3,7-nonadienyl)hydroxyphosphoryl]propyl
Phosphonate (16)--
Butyl lithium (2.85 ml of 1.6 M hexane solution, 4.56 mmol) was added dropwise to a
stirred solution of diethyl
1-[ethoxy(methyl)phosphinoyl]-1-ethylpropylphosphonate 15 (25) (1.200 g, 3.81 mmol) in anhydrous tetrahydrofuran (15 ml) and
cooled at 78 °C under an argon atmosphere. After stirring for
1 h at
78 °C,
,E-geranyl bromide
14 (0.990 g, 4.56 mmol) was added dropwise and the mixture
was stirred for an additional 1 h at
78 °C. The reaction was
quenched with acetic acid (0.546 g, 9.12 mmol), diluted with
CH2Cl2 (60 ml), and washed with brine. The
organic phase was dried and evaporated under reduced pressure. The
residue was purified by silica gel column chromatography, eluting with
ethyl acetate-hexane (1:3). The appropriate fractions were combined,
evaporated, and pump-dried to give the intermediate 16 (0.617 g, 36%) as an oil: 1H NMR (CDCl3):
0.85-1.46 (m, 15H, 5 × CH3CH2), 1.59 (s, 3H, CH3), 1.61 (s, 3H, CH3), 1.69 (s, 3H,
CH3), 1.82-2.36 (m, 12H, 6 × CH2),
3.92-4.35 (m, 6H, 3 × CH3CH2), 4.98-5.19 (m, 2H, 2 × CH); MS (FAB+) m/e 451 (M+H)+.
Tripotassium Salt of
(E)-1-Ethyl-1-[(4,8-dimethyl-3,7-nonadienyl)hydroxyphosphoryl]propyl
Phosphonic Acid (4)--
Compound 4 was
prepared following the experimental procedure reported for
3. 4 (0.351 g, 61%) 1H NMR
(D2O): 0.92 (t, 6H, J = 7.2 Hz, 2 × CH3CH2), 1.48 (s, 3H,
CH3), 1.50 (s, 3H, CH3), 1.54 (s, 3H,
CH3), 1.59-2.15 (m, 12H, 6 × CH2),
5.02-5.18 (m, 2H, 2 × CH); MS (FAB+)
m/e 481 (M+H)+.
Purification of Z-FPP Synthase
Open reading frame Rv1086 (26) was initially cloned into a commercially available protein expression vector pTBY2 (New England BioLabs). However, due to the apparent toxic effects of the Rv1086 fusion protein to the E. coli cells harboring the plasmid, the coding sequence that encoded the complete insert/fusion was moved into a mycobacterial expression vector for expression in M. smegmatis. The following primers were designed to amplify open reading frame Rv1086 from M. tuberculosis H37Rv genomic DNA: 5'-GGTACATATGGAGATCATCCCCCCGCG-3' and 5'-GTCCTGCGCTAGGGCCCCCTGCCGTAGCTG-3'. NdeI and SmaI restriction endonuclease sites were engineered into the above primer sequences respectively (underlined). The native stop codon was removed from the C-terminal primer to allow for an in-frame fusion with the intein/chitin binding domain coded in the vector pTBY2. PCR was performed on a PerkinElmer Life Sciences GeneAmp 2400 PCR system using Vent DNA Polymerase (New England BioLabs). The PCR products were digested with the restriction endonucleases NdeI and SmaI, then ligated into pTBY2, which had been previously digested with the same enzymes. The ligation mixture was electroporated into E. coli-competent cells (XL-1 Blue, Stratagene). Cells containing plasmid were selected on LB agar (EM Science) containing ampicillin at a concentration of 100 µg/ml. Purified plasmid was subjected to restriction and sequence analysis. The resulting construct, pTBY2-Rv1086, was digested with the restriction endonucleases, NdeI and PstI (Life Technologies). The ~2200-bp fragment contained the entire coding sequence for the fusion/insert. This was ligated into pVV16 (a gift from Dr. Varalakshmi Vissa, Colorado State University) and the resulting construct, named pIMP-Rv1086, was electroporated into competent M. smegmatis at 2.5 V, 800 ohms, 25 microfarads. Cells were allowed to recover in LB broth for 90 min and were plated on LB agar with kanamycin (20 µg/ml). A single colony was chosen to start a liquid culture in LB broth with kanamycin (20 µg/ml). The recombinant M. smegmatis strain containing vector pIMP-Rv1086 was grown to mid-log phase in LB broth with 20 µg/ml kanamycin. This genetic construct expressed the M. tuberculosis open reading frame Rv1086 with a C-terminal fusion to a chitin binding domain. Approximately 6 g (wet weight) of cells was harvested by centrifugation, washed with a 0.9% saline solution, and centrifuged again. The resulting pellet was resuspended in cell lysis and column buffer containing 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 0.1 mM EDTA, and 0.1% Triton X-100. The cells were disrupted by probe sonication on ice with a Sanyo Soniprep 150 sonicator (10 cycles of 60 s on and 90 s off), and the suspension was centrifuged at 20,000 × g for 20 min. The pellet was discarded, and the supernatant was loaded onto a 0.8-ml column of chitin beads (New England BioLabs) that had been equilibrated with the same buffer. The expressed protein was eluted from the column essentially as described by the manufacturer, except that the cleavage buffer contained 20 mM Tris-HCl (pH 8.0), 0.1% Triton, 150 mM NaCl, 30 mM dithiothreitol, and 10% glycerol. The eluted fractions were assayed for Z-FPP synthase activity, and the protein concentration was estimated using a BCA protein assay kit (Pierce).
Enzymatic Assays and Product Characterization-- In vitro Z-FPP synthase assays, the enzymatic treatment of reaction products, and the analysis of products were done as described previously (9, 27). Assays were conducted under conditions that were linear for time and protein concentration. In the metal ion dependence studies, the majority of the endogenous divalent cations were removed by incubating the enzyme preparation with Bio-Rex 70 200-400 mesh (sodium form, Bio-Rad) on ice for 20 min. The enzymatic activity was reduced to a basal level, but it was not completely abolished, indicating that residual divalent cations were present. To determine which divalent cations supported activity, CaCl2, MgCl2, MnCl2, or ZnCl2 were added to the assay mixtures at the indicated concentrations. In a separate experiment, 10 mM EDTA was added. To study the pH dependence of Z-FPP synthase activity, a broad-range buffer comprising 250 mM Tris-HCl, 125 mM MES, and 125 mM acetic acid was used. The pH was adjusted with tetraethylammonium hydroxide.
Preparation of Sus scrofa E-FPP Synthase--
Pig brain cytosol
was prepared by homogenizing pig gray matter in 10 mM HEPES
(pH 7.4) and 0.25 M sucrose using 18 passes of a Dounce
homogenizer. The homogenates were centrifuged at 9000 × g for 15 min at 4 °C. The supernatant was decanted and
centrifuged at 142,000 × g for 1 h. The
supernatant was decanted, divided into 1-ml aliquots, stored at
70 °C, and used as an enzyme source for the
,E,E-FPP synthase assays. The
protein concentration was estimated with a BCA protein assay kit (Pierce).
Other Materials--
The sources of all materials have been
described previously (9) with some exceptions. Citronellyl diphosphate,
,E,E-farnesyl diphosphate, and
,E-geranyl diphosphate were synthesized as
described by Davisson et al. (28). Authentic prenols and
prenyl phosphates of various chain lengths were purchased from the
Institute of Biochemistry and Biophysics, Polish Academy of Sciences
(Warsaw, Poland).
,Z-Neryl diphosphate was a
gift from Drs. J. S. Rush and C. J. Waechter (University of Kentucky).
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Purification of Z-FPP Synthase--
Z-FPP synthase was
expressed in M. smegmatis as a fusion protein with a
C-terminal chitin binding domain. Affinity chromatography on a chitin
column permitted a simple one-step protein purification (New England
BioLabs). After the cells were harvested and disrupted, the 20,000 × g supernatant was applied to the chitin column and washed. Subsequent elution with a dithiothreitol-containing buffer cleaved the Z-FPP synthase from the chitin binding domain
via an internal intein site. The column eluate was collected and
subjected to SDS-polyacrylamide gel electrophoresis analysis (Fig.
1). The eluate was also assayed for
[14C]IPP incorporation into butanol-extractable material
with five different allylic primers, DMAPP (C5),
,E-GPP (C10),
,Z-neryl diphosphate
(
,Z-NPP; C10),
,E,E-FPP (C15), and
,E,E,E-GGPP (C20).
,E-GPP and
,Z-NPP were the only functional substrates (data not shown). Assays with the other allylic primers did not produce
any detectable radioactive product. [14C]IPP
incorporation into butanol-extractable material in assays primed with
,E-GPP or
,Z-NPP was
linear for at least 40 min (data not shown). The product of the
,E-GPP assay was analyzed for chain length and
stereochemistry by TLC (Fig. 2),
confirming that the protein fraction shown in Fig. 1 (lane
4) synthesized
,E,Z-FPP. The
product of the
,Z-NPP assay was also analyzed by TLC, demonstrating the enzyme's ability to synthesize a
C15 molecule, presumably
,Z,Z-FPP (data not shown).
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Z-FPP Synthase Reaction Requirements--
Z-FPP
synthase was absolutely dependent on the presence of divalent cation
for activity, and addition of 10 mM EDTA abolished the
enzymatic activity (Table I).
Z-FPP synthase activity was supported by the addition of
MgCl2 or MnCl2. The optimal concentration of
MgCl2 and MnCl2 fell between 0.01 and 1.0 mM. Higher concentrations of MgCl2 (between 1.0 and 5.0 mM) reduced (with respect to optimal concentration)
the activity, whereas high concentrations of MnCl2 (5.0 mM) strongly inhibited it. The enzyme was not stimulated by
ZnCl2 and CaCl2 at the concentrations tested
(0.01-5 mM). Z-FPP synthase was also tested for
optimal activity over a range of pH (5.5-9.5 in 0.5 increments). The
enzyme had a broad peak of activity over pH 7- 8 (data not shown). When
the rate of Z-FPP synthesis was measured in the presence of
saturating ,E-GPP and varying concentrations
of [14C]IPP (Fig.
3A), a Km
value of 124 µM was calculated for the isoprenyl donor by
nonlinear regression (Table II). When the
concentration of IPP was fixed and the concentration of
,E-GPP or
,Z-NPP was
varied, Michaelis constants of 38 and 16 µM were calculated (Fig. 3 and Table II).
|
|
|
Inhibition of Z-FPP Synthase--
Compounds 1-4
(Scheme 1) and citronellyl diphosphate (Cit-PP, Fig.
4A) were tested for the
ability to inhibit the synthesis of
,E,Z-FPP by the Z-FPP
synthase from M. tuberculosis and the synthesis of
,E,E-FPP by the E-FPP
synthase from pig brain (S. scrofa). Z-FPP
synthase was inhibited by compound 4 and Cit-PP (Fig.
4B). The respective IC50 values were estimated
to be 300 and 350 µM in the presence of 100 µM GPP. E-FPP synthase was inhibited only by
Cit-PP with an IC50 of 125 µM under similar assay conditions (Fig. 4C). Compounds 1, 2, and 3 had no effect on either of the FPP
synthases at the concentrations tested (data not shown).
|
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DISCUSSION |
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Little is known about Z-isoprenyl diphosphate synthases. Some early partial purification and characterization work has been published for bacterial (29, 30) and eukaryotic (31-35) Z-isoprenyl diphosphate synthases, but it was not until Shimizu et al. (36) purified undecaprenyl diphosphate synthase from M. luteus in 1998 that a gene and protein sequence were correlated with the biochemical data. The complete sequencing of the M. tuberculosis H37Rv genome (and other genomes) has since revealed a wealth of information. Apfel et al. (37) was able to find 28 homologs of M. luteus undecaprenyl diphosphate synthase using comparative genome queries. M. tuberculosis is unique among organisms that have had their genomes sequenced in that there are two genes with homology to Z-isoprenyl diphosphate synthases (Rv1086 and Rv2361c), as opposed to the single Z-isoprenyl diphosphate synthase homolog typically found in the genomes of other organisms (37). We previously identified the function of the enzymes encoded by Rv1086 (Z-FPP synthase) and Rv2361c (Z-decaprenyl diphosphate synthase) (9). In this study, we report the purification, characterization, and inhibition of Rv1086.
All isoprenyl diphosphate synthases (E and Z)
studied thus far have a strict requirement for a divalent cation.
However, the type of cation and the concentration required for optimal
activity is unique to each enzyme. X-ray crystallography of avian
E-FPP synthase revealed that Mg2+ cations were
positioned within the active site of the enzyme (bound to aspartate
residues) while complexed with the diphosphate moiety of the substrate
(18). Undecaprenyl diphosphate synthase from Lactobacillus
plantarum and E. coli were both shown to require Mg2+ ions to bind IPP and a radiolabeled photolabile analog
of ,E,E-FPP (38). The mycobacterial
Z-FPP synthase is no exception, because it is stimulated by
both Mg2+ and Mn2+. It is likely that the
divalent cation is required for substrate binding in a similar
mechanism to that seen in E-isoprenyl diphosphate synthases.
Studies have shown that many E-FPP synthases can accept both
DMAPP and ,E-GPP as allylic substrates
(39-43). When synthesizing
,E,E-FPP from DMAPP, the enzyme
completes two condensation reactions with IPP, releasing only trace
amounts of the intermediate
,E-GPP (3). In
contrast, DMAPP was not a functional substrate for the Z-FPP
synthase.
,E-GPP and
,Z-NPP were the only effective allylic
substrates tested, supporting the synthesis of
,E,Z-FPP and
,Z,Z-FPP, respectively. Although
little is known about the intracellular concentrations of IPP or the
allylic substrates,
,E-GPP or
,Z-NPP, in M. tuberculosis, the
observed Michaelis constants for these substrates are 124, 38, and 16 µM, respectively. Long-chain Z-isoprenyl
diphosphate synthases have been shown to be capable of utilizing
allylic primers with different stereochemistries as substrates (3, 44).
If the Pol-P from M. tuberculosis is structurally similar to
the Pol-P from M. smegmatis
(
,E,polyZ-decaprenyl phosphate),
,E-GPP is the natural substrate of
Z-FPP synthase. It is possible that
,Z-NPP is a precursor to an, as yet,
undescribed isoprenoid molecule in M. tuberculosis.
Nevertheless, the Z-FPP synthase is a monofunctional enzyme,
and a separate enzyme must exist in M. tuberculosis that
synthesizes either
,E-GPP or
,Z-NPP from DMAPP and IPP.
A comparison of the mycobacterial Z-FPP synthase with the
E-FPP synthase from S. scrofa brain was of
interest, because the two enzymes catalyze the addition of IPP to
,E-GPP but release products with opposite
stereochemistry at the newly formed double bond. Substrate analogs such
as Cit-PP have been previously shown to inhibit pig liver
E-FPP synthase (45, 46). Cit-PP and other substrate analogs
are thought to bind the enzyme active site through nonspecific
lipophilic forces and a diphosphate binding force (45). The enzyme
activity is inhibited, because Cit-PP lacks the allylic double bond in
the 2 position (Fig. 4A) and is not able to undergo the
typical electrophilic alkylation reaction. As shown in Fig. 4
(B and C), Cit-PP was effective at inhibiting both the E- and the Z-FPP synthases.
Compounds 1-4 (Scheme 1) were designed as analogs of
,E-GPP in which the biologically labile
diphosphate moiety was replaced by moieties that can act as stable
isosters that possess different conformational and stereoelectronic
characteristics such as an unsubstituted phosphonoacetamidoxy (compound
1), an
-ethyl-substituted phosphonoacetamidoxy (compound
2), a phosphonoacetamido group (compound 3), or
an
,
'-diethyl-substituted [methylen(hydroxy)phosphoryl]methanphosphonic moiety (compound 4). These isosteric moieties were chosen bearing in mind previous studies on isosters of the diphosphate group of prenyl diphosphates as squalene synthase (25), protein:farnesyl transferase (47), and protein:geranylgeranyl transferase inhibitors (48, 49).
Compounds 1-3 had no effect on either the Z-FPP synthase or the E-FPP synthase (data not shown). Compound
4 specifically inhibited the Z-FPP synthase (Fig.
4B) but had no effect on the activity of the
E-FPP synthase (Fig. 4C). Despite its relatively
high IC50, the
,
'-diethyl-substituted
[methylen(hydroxy)phosphoryl]methanphosphonic moiety of compound
4 seems to possess the correct conformational and
stereoelectronic features to selectively interact with the active site
of the mycobacterial Z-FPP synthase, suggesting that the
study of the active site of this previously uncharacterized enzyme
could lead to the development of novel chemotherapeutic agents for the
treatment of multiple drug-resistant tuberculosis.
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FOOTNOTES |
---|
* This work was supported by Grant AI18357 from NIAID, National Institutes of Health and Program Project AI46393 from the National Cooperative Drug Discovery Group, Opportunistic Infections in AIDS, NIAID, NIH.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Tel.: 970-491-3308; Fax: 970-491-1815; E-mail dcrick@cvmbs.colostate.edu.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M007168200
2
The stereochemical configuration of the isoprene
units is always listed starting at the omega () end of the molecule.
3 M. C. Schulbach, and D. C. Crick, unpublished.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
IPP, isopentenyl
diphosphate;
Cit-PP, citronellyl diphosphate;
DMAPP, dimethylallyl
diphosphate;
, E-GPP,
,E-geranyl diphosphate;
, E,E,E-GGPP,
,E,E,E-geranylgeranyl
diphosphate;
, E,Z-FPP,
,E,Z-farnesyl diphosphate;
, E,E-FPP,
,E,E-farnesyl diphosphate;
, Z,Z-FPP,
,Z,Z-farnesyl diphosphate;
Pol-P, polyprenyl phosphate;
Z-FPP synthase,
,E,Z-farnesyl diphosphate synthase;
E-FPP synthase, W,E,E-farnesyl diphosphate synthase;
, Z-NPP,
,Z-neryl
diphosphate;
MS, mass spectrometry;
PCR, polymerase chain reaction;
bp, base pair(s);
MES, 4-morpholineethanesulfonic acid;
MOPS, 4-morpholinepropanesulfonic acid.
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