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Farnesyl-diphosphate synthase (FPPS) catalyzes the synthesis of farnesyl diphosphate, an important precursor of sterols, dolichols, ubiquinones, and prenylated proteins. We report the cloning and characterization of two Toxoplasma gondii farnesyl-diphosphate synthase (TgFPPS) homologs. A single genetic locus produces two transcripts, TgFPPS and TgFPPSi, by alternative splicing. Both isoforms were heterologously expressed in Escherichia coli, but only TgFPPS was active. The protein products predicted from the nucleotide sequences have 646 and 605 amino acids and apparent molecular masses of 69.5 and 64.5 kDa, respectively. Several conserved sequence motifs found in other prenyl-diphosphate synthases are present in both TgFPPSs. TgFPPS was also expressed in the baculovirus system and was biochemically characterized. In contrast to the FPPS of other eukaryotic organisms, TgFPPS is bifunctional, catalyzing the formation of both farnesyl diphosphate and geranylgeranyl diphosphate. TgFPPS localizes to the mitochondria, as determined by the co-localisation of the affinity-purified antibodies against the protein with MitoTracker, and in accord with the presence of an N-terminal mitochondria-targeting signal in the protein. This enzyme is an attractive target for drug development, because the order of inhibition of the enzyme by a number of bisphosphonates is the same as that for inhibition of parasite growth. In summary, we report the first bifunctional farnesyl-diphosphate/geranylgeranyl-diphosphate synthase identified in eukaryotes, which, together with previous results, establishes this enzyme as a valid target for the chemotherapy of toxoplasmosis.

Trypanosoma cruzi, the etiologic agent of Chagas disease, has an unusual ATP-dependent hexokinase (TcHK) that is not affected by D-glucose 6-phosphate, but is non-competitively inhibited by inorganic pyrophosphate (PP(i)), suggesting a heterotropic modulator effect. In a previous study we identified a novel family of bisphosphonates, metabolically stable analogs of PP(i), which are potent and selective inhibitors of TcHK as well as the proliferation of the clinically relevant intracellular amastigote form of the parasite in vitro (Hudock, M. P., Sanz-Rodriguez, C. E., Song, Y., Chan, J. M., Zhang, Y., Odeh, S., Kosztowski, T., Leon-Rossell, A., Concepcion, J. L., Yardley, V., Croft, S. L., Urbina, J. A., and Oldfield, E. (2006) J. Med. Chem. 49, 215-223). In this work, we report a detailed kinetic analysis of the effects of three of these bisphosphonates on homogeneous TcHK, as well as on the enzyme in purified intact glycosomes, peroxisome-like organelles that contain most of the glycolytic pathway enzymes in this organism. We also investigated the effects of the same compounds on glucose consumption by intact and digitonin-permeabilized T. cruzi epimastigotes, and on the growth of such cells in liver-infusion tryptose medium. The bisphosphonates investigated were several orders of magnitude more active than PP(i) as non-competitive or mixed inhibitors of TcHK and blocked the use of glucose by the epimastigotes, inducing a metabolic shift toward the use of amino acids as carbon and energy sources. Furthermore, there was a significant correlation between the IC(50) values for TcHK inhibition and those for epimastigote growth inhibition for the 12 most potent compounds of this series. Finally, these bisphosphonates did not affect the sterol composition of the treated cells, indicating that they do not act as inhibitors of farnesyl diphosphate synthase. Taken together, our results suggest that these novel bisphosphonates act primarily as specific inhibitors of TcHK and may represent a novel class of selective anti-T. cruzi agents.

Inorganic polyphosphate (polyP) has been identified and measured in human platelets. Millimolar levels (in terms of Pi residues) of short chain polyP were found. The presence of polyP of approximately 70-75 phosphate units was identified by 31P NMR and by urea-polyacrylamide gel electrophoresis of platelet extracts. An analysis of human platelet dense granules, purified using metrizamide gradient centrifugation, indicated that polyP was preferentially located in these organelles. This was confirmed by visualization of polyP in the dense granules using 4',6-diamidino-2-phenylindole and by its release together with pyrophosphate and serotonin upon thrombin stimulation of intact platelets. Dense granules were also shown to contain large amounts of calcium and potassium and both bafilomycin A1-sensitive ATPase and pyrophosphatase activities. In agreement with these results, when human platelets were loaded with the fluorescent calcium indicator Fura-2 acetoxymethyl ester to measure their intracellular Ca2+ concentration ([Ca2+]i), they were shown to possess a significant amount of Ca2+ stored in an acidic compartment. This was indicated by the following: 1) the increase in [Ca2+]i induced by nigericin, monensin, or the weak base, NH4Cl, in the nominal absence of extracellular Ca2 and 2) the effect of ionomycin, which could not take Ca2+ out of acidic organelles and was more effective after alkalinization of this compartment by the previous addition of nigericin, monensin, or NH4Cl. All of these characteristics of the platelet dense granules, together with their known acidity and high density (both by weight and by electron microscopy), are similar to those of acidocalcisomes (volutin granules, polyP bodies) of bacteria and unicellular eukaryotes. The results suggest that acidocalcisomes have been conserved during evolution from bacteria to humans.

We report the cloning and sequencing of a gene encoding the farnesyl pyrophosphate synthase (FPPS) of Trypanosoma brucei. The protein (TbFPPS) is an attractive target for drug development because the growth of T. brucei has been shown to be inhibited by analogs of its substrates, the nitrogen containing bisphosphonates currently in use in bone resorption therapy. The protein predicted from the nucleotide sequence of the gene has 367 amino acids and a molecular mass of 42 kDa. Several sequence motifs found in other FPPSs are present in TbFPPS, including an 11-mer peptide insertion present also in the Trypanosoma cruzi FPPS. Heterologous expression of TbFPPS in Escherichia coli produced a functional enzyme that was inhibited by several nitrogencontaining bisphosphonates, such as pamidronate and risedronate. Risedronate was active in vivo against T. brucei infection in mice (giving a 60% survival rate), but pamidronate was not effective. The essential nature of TbFPPS was studied using RNA interference (RNAi) to inhibit the expression of the gene. Expression of TbFPPS double-stranded RNA in procyclic trypomastigotes caused specific degradation of mRNA. After 4 days of RNAi, the parasite growth rate declined and the cells subsequently died. Similar results were obtained with bloodstream form trypomastigotes, except that the RNAi system in this case was leaky and mRNA levels and parasites recovered with time. Molecular modeling and structure-activity investigations of enzyme and in vitro growth inhibition data resulted in similar pharmacophores, further validating TbFPPS as the target for bisphosphonates. These results establish that FPPS is essential for parasite viability and validate this enzyme as a target for drug development.

We report the cloning and sequencing of a gene encoding the farnesyl pyrophosphate synthase of Trypanosoma cruzi. The protein (T. cruzi farnesyl pyrophosphate synthase, TcFPPS) is an attractive target for drug development, since the growth of T. cruzi is inhibited by carbocation transition state/reactive intermediate analogs of its substrates, the nitrogen-containing bisphosphonates currently in use in bone resorption therapy. The protein predicted from the nucleotide sequence of the gene has 362 amino acids and a molecular mass of 41.2 kDa. Several sequence motifs found in other FPPSs are present in TcFPPS. Heterologous expression of TcFPPS in Escherichia coli produced a functional enzyme that was inhibited by the nitrogen-containing bisphosphonates alendronate, pamidronate, homorisedronate, and risedronate but was less sensitive to the non-nitrogen-containing bisphosphonate etidronate, which, unlike the nitrogen-containing bisphosphonates, does not affect parasite growth. The protein contains a unique 11-mer insertion located near the active site, together with other sequence differences that may facilitate the development of novel anti-Chagasic agents.

High field (31)P nuclear magnetic resonance spectroscopy showed that inorganic pyrophosphate (P(2)O(7)(4-)) is more abundant than ATP in Trypanosoma cruzi, the causative agents of Chagas' disease. These results were confirmed by specific analytical assays, which showed that in epimastigotes, the concentrations of inorganic pyrophosphate and ATP were 194.7 +/- 25.9 and 37.6 +/- 5.5 nmol/mg of protein, respectively, and for the amastigote form, the corresponding concentrations were 358.0 +/- 17.0 and 36.0 +/- 1.9 nmol/mg of protein. High performance liquid chromatographic analysis of perchloric acid extracts of epimastigotes labeled for 3 h with (32)P-orthophosphate showed a significant incorporation of the precursor into inorganic pyrophosphate. Inorganic pyrophosphate was not uniformly distributed in T. cruzi but was shown by (31)P-NMR and chemical analysis to be particularly associated with acidocalcisomes, organelles shown previously to contain large amounts of phosphorus and various elements. Electron microscopy analysis of pyrophosphatase-treated permeabilized epimastigotes showed disappearance of the electron density of the acidocalcisomes. Nonmetabolizable analogs of pyrophosphate, currently used for the treatment of bone resorption disorders, selectively inhibited the proliferation of intracellular T. cruzi amastigotes and produced a profound suppression in the number of circulating trypomastigotes in mice with an acute infection of T. cruzi, offering a potentially new route to chemotherapy.

Deuterium nuclear magnetic resonance spectra (at 55.3 MHz) have been obtained of 19F-2H double-labeled phospholipids in pure lipid bilayers, and of 2H-labeled lipid in a 19F-labeled bilayer, as a function of concentration, to assess the perturbing influence of 19F sites in lipid hydrocarbon chains. Order parameters of 2H-labeled sites adjacent to C-8 myristic fluorine probes in pure lipid bilayers, and 19F spin label order parameters themselves, are about 30% lower than those deduced from the use of nonperturbing 2H probes. The effect is intramolecular rather than intermolecular and presumably represents increased gauche states due to the increased size of the 19F label. This effect is consistent with the view that difluoromethylene fatty acyl chains function in a manner approximating that of unsaturated fatty acyl chains. The differences disappear in the presence of cholesterol at very high order parameters (Smol approximately 0.8 to 0.9). These results represent the first attempt at elucidating the perturbing effects of a high sensitivity probe (19F) and indicate that caution must be used when using spectroscopic probes to deduce the absolute magnitude of hydrocarbon chain order parameters.

Natural abundance 13C Fourier transform NMR spectra (at 15.18 MHz, in 20-mm sample tubes) of aqueous native proteins yield numerous narrow single carbon resonances of nonprotonated aromatic carbons. Techniques for the assignment of these resonances are presented. Each technique is applied to one or more of the following proteins: ferricytochrome c from horse heart and Candida krusei, ferrocytochrome c and cyanoferricytochrome c from horse heart, lysozyme from hen egg white, cyanoferrimyoglobins from horse and sperm whale skeletal muscle, and carbon monoxide myoglobin from horse. In all of the protein spectra we have examined, methine aromatic carbons give rise to broad bands. Studies of the narrow resonances of nonprotonated aromatic carbons of proteins are facilitated by removal of these broad bands by means of the convolution-difference method, preferably from spectra recorded under conditions of noise-modulated off-resonance proton decoupling. We present a summary of the chemical shift ranges for the various types of nonprotonated aromatic carbons of amino acid residues and hemes of diamagnetic proteins, based on our results for hen egg white lysozyme, horse heart ferrocytochrome c, horse carbon monoxide myoglobin, and carbon monoxide hemoglobins from various species...

We report the observation of deuterium Fourier transform NMR spectra (obtained by the quadrupole echo method at 8.5 Tesla, corresponding to a 2H resonance frequency of 55.3 MHz) of [meso-alpha, beta, gamma, delta-2H4], [methyl-1,3-2H6], and [methylene-6,7-b-2H4]heme-labeled aquoferrimyoglobin microcrystals (in approximately 90% saturated (NH4)2SO4 at pH 6.8) from Physeter catodon, using the method of magnetic ordering (Rothgeb, T. M. and Oldfield, E. (1981) J. Biol. Chem. 256, 1432-1446). The results, together with those obtained on suitable diamagnetic derivatives, permit partial determination of the static organization of the heme, and the results obtained are in good agreement with those obtained using x-ray crystallography (Takano, T. (1977( J. Mol. Biol. 110, 537-568). We show that resonances near the paramagnetic iron center are subject to extremely large (approximately 500 ppm) hyperfine shifts, which distort the otherwise symmetric 2H spectra. Temperature dependence studies are required to analyze these shifts, which are an order of magnitude larger than those seen in solution NMR spectroscopy. The overall results suggest that 2H solid state NMR spectroscopy of magnetically ordered paramagnetic protein microcrystals may be a useful method for determination of heme organization in systems that for one reason or another are unsuitable for analysis using x-ray diffraction methods.

Proton-decoupled natural abundance 13C NMR spectra of carbon monoxide hemoglobins were recorded at 15.18 MHz by the Fourier transform method, under conditions of spectrometer sensitivity sufficient for detection of individual carbon resonances. The aromatic region of each spectrum contains broad bands of methine carbon resonances, and some relatively narrow peaks arising from nonprotonated carbons. Resonances of heme carbons were detected in spectra of carbon monoxide hemoglobins, but not in spectra of ferrihemoglobin (as a result of paramagnetic effects). Spectra of carbon monoxide hemoglobins from various species yielded only a few well resolved individual carbon resonances, most notably those of Cgamma of tryptophan residues. A comparison of the spectra of human adult, human fetal, chicken AII, and bovine fetal hemoglobins yielded specific assignments for all resonances of Cgamma of tryptophan residues. In the cases of human fetal, chicken AII, and bovine fetal hemoglobins, each tryptophan yielded a completely resolved individual carbon resonance. The chemical shift difference between the resonances of Cgamma of Trp-130beta and Cgamma of Trp-37beta is about 6 ppm. The chemical shift difference between Trp A12[14]alpha and Trp A12[15]beta is 1 ppm or less. A comparison of the chemical shifts of analogous tryptophan residues of the four carbon monoxide hemoglobins suggests very similar conformations in solution.

We have obtained oxygen-17 (17O) nuclear magnetic resonance (NMR) spectra of C17O ligands bound to ferrous horseradish peroxidase isozyme A, isozyme C, and ferrous chloroperoxidase, as a function of pH. Our results show that the peroxidases exist in two distinct states, the acidic and alkaline forms, which undergo reversible acid-base-induced transitions characterized by a single pK value. The two forms are characterized spectroscopically in much the same way in all three proteins, suggesting a similar structural origin for the transition process. In particular, the 17O NMR signal of the acidic form is approximately 7 ppm more shielded than that of the alkaline form, and the CO ligand in the acidic form appears to have a smaller 17O nuclear quadrupole coupling constant than that of the alkaline form. We have also obtained the pK values and exchange rates for all three peroxidases. The results indicate that a similar structural change may be involved in the transition process in all three peroxidases.

We have obtained the first deuterium NMR spectra of an individual membrane protein, bacteriorhodopsin in the purple membrane of Halobacterium halobium R1. Biosynthetic isotopic enrichment with [gamma-2H6]valine and high field Fourier transform operation permitted rapid data acquisition on intact membranes, including measurement of relaxation times. At some temperatures high quality spectra could be obtained in less than 1 s. [U-14C]Valine tracer studies indicate that less than or equal to 2% of valine added to the growth medium is broken down and incorporated into other membrane constituents. The NMR results indicate that the valine side chain is a rather rigid structure. Motion about C alpha-C beta is slow (less than 10(5) s-1) at growth temperature, While motion about C beta-C gamma is as expected fast (much greater than 10(5) s-1) at all accessible temperatures. The activation energy for methyl group rotation from spin-lattice relaxation data between -75 and 53 degrees C is approximately 2.4 kcal/mol, in good agreement with previous 1H NMR studies on solid alkanes. Preliminary data on [gamma-2H6]valine-labeled Acholeplasma laidlawii B (PG9) cell membranes are also presented. Our results strongly suggest that it should now be possible to observe in great detail the motions of any type of amino acid side chain in membrane proteins, including the effects of lipid composition on protein dynamics.

A new technique capable of determining the static and dynamic structures of heme protein crystals is reported. It is shown that microcrystals of a variety of paramagnetic heme proteins, suspended in approximately 90% saturated (NH4)2SO4, may be perfectly aligned by an intense static external magnetic field, H0, due to the large anisotropy in the magnetic susceptibility of the protein caused by the paramagnetic center. Myoglobin from sperm whale (Physeter catodon) was isotopically enriched at the C epsilon methyl groups of methionine residues 55 and 131 with either 13C or 2H and studied in the crystalline solid state by 2H-quadrupole echo and 13C-Fourier transform nuclear magnetic resonance spectroscopy. It was found that suspensions of both high (S = 5/2) and low (S = 1/2) spin ferric forms of the labeled protein were ordered, the axis of ordering being approximately perpendicular to the low temperature minimum g tensor valve, even though upper Kramers levels are populated at room temperature. The paramagnetic CoII derivative "coboglobin" showed similar ordering behavior, but the diamagnetic carboxymyoglobin was unaffected. The magnetic ordering method permits the recording of "single crystal" NMR spectra from microcrystalline arrays of proteins which cannot be prepared in large enough form (approximately 1 cm3) for single crystal NMR spectroscopy and thereby allows the resolution and assignment of numerous single atom sites in the crystalline solid state. The information from a "single crystal" NMR spectrum combined with that obtained on the crystal powder allows for the direct determination of (i) the spatial orientation of the particular labeled residue within the protein crystal and (ii) the rates and types of side chain motion. Resonances were assigned by spin label broadening experiments and by use of existing x-ray data to predict 2H-NMR spectra. This new technique opens up the possibility of determining directly the dynamic structure of protein crystals and of comparing the structures of proteins in the crystalline solid state with that in solution and is applicable to other heme proteins, e.g. catalase.

The aromatic regions in proton-decoupled natural abundance 13C Fourier transform nuclear magnetic resonance spectra (at 14.2 kG) of small native proteins contain broad methine carbon bands and narrow nonprotonated carbon resonances. Some factors that affect the use of natural abundance 13C Fourier transform NMR spectroscopy for monitoring individual nonprotonated aromatic carbon sites of native proteins in solution are discussed. The effect of protein size is evaluated by comparing the 13C NMR spectra of horse heart ferrocytochrome c, hen egg white lysozyme, horse carbon monoxide myoglobin, and human adult carbon monoxide hemoglobin. Numerous single carbon resonances are observed in the aromatic regions of 13C NMR spectra of cytochrome c, lysozyme, and myoglobin. The much larger hemoglobin yields few resolved individual carbon resonances. Theoretical and some experimental values are presented for the natural linewidths (W), spin-lattice relaxation times (T1), and nuclear Overhauser enhancements (NOE) of nonprotonated aromatic carbons and Czeta of arginine residues. In general, the 13C-1H dipolar mechanism dominates the relaxation of these carbons. 13C-14N dipolar relaxation contributes significantly to 1/T1 of C epsilon2 of tryptophan residues and Czeta of arginine residues of proteins in D2O. The NOE of each nonprotonated aromatic carbon is within experimental error of the calculated value of about 1.2. As a result, integrated intensities can be used for making a carbon count. Theoretical results are presented for the effect of internal rotation on W, T1, and the NOE. A comparison with the experimental T1 and NOE values indicates that if there is internal rotation of aromatic amino acid side chains, it is not fast relative to the over-all rotational motion of the protein.