Purification, morphometric analysis, and characterization of the glycosomes (microbodies) of the protozoan hemoflagellate Trypanosoma brucei.

Trypanosoma brucei glycosomes (microbodies containing nine enzymes involved in glycolysis) have been purified to near homogeneity from bloodstream-form trypomastigotes for the purpose of morphologic and biochemical analysis. Differential centrifugation followed by two isopycnic centrifugations in an isotonic Percoll and in a sucrose gradient, respectively, resulted in 12- to 13-fold purified glycosomes with an overall yield of 31%. These glycosomes appeared to be highly pure and contained less than 1% mitochondrial contamination as judged by morphometric and biochemical analyses. In intact cells, glycosomes displayed a remarkably homogeneous size distribution centered on an average diameter of 0.27 micron with a standard deviation of 0.03 micron. The size distribution of isolated glycosomes differed only slightly from that measured in intact cells. One T. brucei cell contained on average 230 glycosomes, representing 4.3% of the total cell volume. The glycosomes were surrounded by a single membrane and contained as phospholipids only phosphatidyl choline and phosphatidyl ethanolamine in a ratio of 2:1. The purified glycosomal fraction had a very low DNA content of 0.18 microgram/mg protein. No DNA molecules were observed that could not have been derived from contaminating mitochondrial or nuclear debris.

A potential target enzyme for trypanocidal drugs revealed by the crystal structure of NAD-dependent glycerol-3-phosphate dehydrogenase from Leishmania mexicana.

BACKGROUND: NAD-dependent glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the interconversion of dihydroxyacetone phosphate and L-glycerol-3-phosphate. Although the enzyme has been characterized and cloned from a number of sources, until now no three-dimensional structure has been determined for this enzyme. Although the utility of this enzyme as a drug target against Leishmania mexicana is yet to be established, the critical role played by GPDH in the long slender bloodstream form of the related kinetoplastid Trypanosoma brucei makes it a viable drug target against sleeping sickness. RESULTS: The 1.75 A crystal structure of apo GPDH from L. mexicana was determined by multiwavelength anomalous diffraction (MAD) techniques, and used to solve the 2.8 A holo structure in complex with NADH. Each 39 kDa subunit of the dimeric enzyme contains a 189-residue N-terminal NAD-binding domain and a 156-residue C-terminal substrate-binding domain. Significant parts of both domains share structural similarity with plant acetohydroxyacid isomeroreductase. The discovery of extra, fatty-acid like, density buried inside the C-terminal domain indicates a possible post-translational modification with an associated biological function. CONCLUSIONS: The crystal structure of GPDH from L. mexicana is the first structure of this enzyme from any source and, in view of the sequence identity of 63%, serves as a valid model for the T. brucei enzyme. The differences between the human and trypanosomal enzymes are extensive, with only 29% sequence identity between the parasite and host enzyme, and support the feasibility of exploiting the NADH-binding site to develop selective inhibitors against trypanosomal GPDH. The structure also offers a plausible explanation for the observed inhibition of the T. brucei enzyme by melarsen oxide, the active form of the trypanocidal drugs melarsoprol and cymelarsan.

Alkyl dihydroxyacetone phosphate synthase in glycosomes of Trypanosoma brucei.

Alkyl-dihydroxyacetone phosphate synthase (E.C. 2.5.1.26), the key enzyme in ether phospholipid biosynthesis, was demonstrated to be present in Trypanosoma brucei. The distribution of alkyl-dihydroxyacetone phosphate synthase was found to be identical to that of dihydroxyacetone phosphate acyltransferase (E.C. 2.3.1.42), which has previously been shown to be exclusively associated with the glycosome fraction (Opperdoes, F.R. (1984) FEBS Lett. 169, 35-39). Studies with gradient purified glycosomes indicated that the formation of alkyl-dihydroxyacetone phosphate was completely dependent on the presence of acyl-dihydroxyacetone phosphate. The glycosomal alkyl-dihydroxyacetone phosphate synthase activity was characterized with respect to its pH optimum, Triton X-100 sensitivity and the dependency on the concentration of the substrates palmitoyl-dihydroxyacetone phosphate and hexadecanol. Using thin-layer chromatographic and alkaline hydrolysis procedures the reaction product was identified as alkyl-dihydroxyacetone phosphate. Alkyl-dihydroxyacetone phosphate synthase was resistant to proteolytic inactivation by trypsin in intact glycosomes but not in Triton X-100 disrupted glycosomes. It is concluded that T. brucei glycosomes contain the enzymes responsible for glycero-ether bond formation analogous to mammalian peroxisomes.

Purification and characterisation of the phosphoglycerate kinase isoenzymes of Trypanosoma brucei expressed in Escherichia coli.

The Trypanosoma brucei phosphoglycerate kinase (PGK) glycosomal and cytosolic isoenzymes have been overexpressed in Escherichia coli and purified to near-homogeneity. Both enzymes were similar to the corresponding natural proteins with respect to their physicochemical and kinetic properties. In addition, a mutant of the glycosomal PGK lacking the 20 amino acid long C-terminal extension was overexpressed and purified. Various properties of this truncated glycosomal PGK were examined and it was found that in some aspects the protein behaved quite differently when compared with its natural counterpart. This was notably the case for the apparent Km for 3-phosphoglyceric acid, its sensitivity to inhibitors and its response to salts and guanidine HCl. However, its Vmax was found to be similar to that of the natural glycosomal PGK. These results suggest that the changes in the C-terminus caused a conformational change effecting the 3-phosphoglyceric acid binding site located at the N-terminal domain of the protein.

Inhibition of the glycolytic enzymes in the trypanosome: an approach in the development of new leads in the therapy of parasitic diseases.

Glycolysis in the trypanosome represents an important target for the development of new therapeutic agents due to the fact that this metabolism is essential for the parasite, glucose being its sole source of energy. In addition, different features of this metabolism and those associated with glycolytic enzymes offer opportunities for the development of efficient and selective compounds. Examples are given in this work of inhibitors directed to the enzymes aldolase and glyceraldehyde-phosphate-dehydrogenase and also of molecules acting specifically on the clusters of basic amino-acids present at the surfaces of the glycolytic enzymes in the parasite.

Preliminary crystallographic studies of triosephosphate isomerase from the blood parasite Trypanosoma brucei brucei.

Crystals of triosephosphate isomerase (EC 5.3.1.1) from Trypanosoma brucei brucei have been grown. These crystals diffract to at least 2 A, even after 60 hours of exposure to X-rays. The space group is P212121, with cell dimensions a = 112.4 A, b = 97.8 A, c = 48.0 A. There is one dimer per asymmetric unit.

Preliminary crystallographic studies of glycosomal glyceraldehyde phosphate dehydrogenase from Trypanosoma brucei brucei.

Crystals of glyceraldehyde phosphate dehydrogenase from the glycosome of Trypanosoma brucei brucei have been grown, and a partial data set has been collected using synchrotron radiation. The crystals diffract initially to 2.3 A resolution. The space group is P2(1)2(1)2, with cell dimensions a = 135 A, b = 255 A, c = 115 A, so there are probably at least two tetramers in the asymmetric unit.

Structure of the complex between trypanosomal triosephosphate isomerase and N-hydroxy-4-phosphono-butanamide: binding at the active site despite an "open" flexible loop conformation.

The structure of triosephosphate isomerase from Trypanosoma brucei complexed with the competitive inhibitor N-hydroxy-4-phosphono-butanamide was determined by X-ray crystallography to a resolution of 2.84 A. Full occupancy binding of the inhibitor is observed only at one of the active sites of the homodimeric enzyme where the flexible loop is locked in a completely open conformation by crystal contacts. There is evidence that the inhibitor also binds to the second active site of the enzyme, but with low occupancy. The hydroxamyl group of the inhibitor forms hydrogen bonds to the side chains of Asn 11, Lys 13, and His 95, whereas each of its three methylene units is involved in nonpolar interactions with the side chain of the flexible loop residue Ile 172. Interactions between the hydroxamyl and the catalytic base Glu 167 are absent. The binding of this phosphonate inhibitor exhibits three unusual features: (1) the flexible loop is open, in contrast with the binding mode observed in eight other complexes between triosephosphate isomerase and various phosphate and phosphonate compounds; (2) compared with these complexes the present structure reveals a 1.5-A shift of the anion-binding site; (3) this is the first phosphonate inhibitor that is not forced by the enzyme into an eclipsed conformation about the P-CH2 bond. The results are discussed with respect to an ongoing drug design project aimed at the selective inhibition of glycolytic enzymes of T. brucei.

Molecular analysis of phosphoglycerate kinase in Trypanoplasma borreli and the evolution of this enzyme in kinetoplastida.

In the protozoan kinetoplastid organism Trypanoplasma borreli, phosphoglycerate kinase (PGK) activity was found in two different cell compartments: 80% in the cytosol and 20% in peroxisome-like organelles called glycosomes. However, only one functional pgk gene could be detected, in addition to a pseudo-pgk gene. No short-range linkage could be established between these two genes, although they are presumably present on the same chromosome. The intact gene codes for a polypeptide of 411 amino acids, with a C-terminal extension of four residues, -VAKF, a sequence with probably a low targeting efficiency for glycosomes. The calculated net charge and molecular mass of the encoded polypeptide are +13 and 44230Da, respectively. In other Kinetoplastida, different tandemly arranged genes code for distinct PGK isoenzymes in glycosomes and cytosol. By comparison of the pgk gene organization, and a phylogenetic analysis, we have traced a plausible scenario of the evolution of the PGK isoenzymes in these organisms and of the enzymes' intracellular compartmentation.

Localization of the initial steps in alkoxyphospholipid biosynthesis in glycosomes (microbodies) of Trypanosoma brucei.

Cell fractionation of Trypanosoma brucei cultured procyclic stages showed that the key enzyme of glycerol-ether lipid synthesis, dihydroxyacetone-phosphate acyltransferase (EC 2.3.1.42) was exclusively associated with the microbody fraction. These organelles contained in addition 1-acyl glycerol-3-phosphate: NADP+ oxidoreductase (EC 1.1.1.101) and acyl-CoA reductase and were capable of utilizing DHAP, but not G-3-P, as substrate for lysophosphatidic acid formation. It is concluded that in T. brucei the glycosomes are the exclusive site of the synthesis of precursors for glycerol-ether lipid synthesis and that they contain the entire pathway to form alkoxylipids from glycerol and acyl-CoA.

Topogenesis of glycolytic enzymes in Trypanosoma brucei.

The protozoan haemoflagellate Trypanosoma brucei, differs from other eukaryotic cells in that it contains nine enzymes involved in glucose and glycerol metabolism which are associated with microbody-like organelles called glycosomes. The information available to date indicates that glycosomal enzymes are synthesized as polypeptides of mature size. For three of them, glyceraldehyde-phosphate dehydrogenase, aldolase and glycerol-3-phosphate dehydrogenase, it has been shown that they are made on free polysomes in the cytosol and are subsequently transferred to the glycosome without any secondary modification. The topogenic signal responsible for import into the glycosome must, therefore, be present in the mature protein. Remarkable differences exist between the latter proteins and other glycolytic enzymes: (i) most glycosomal proteins have an apparent Mr which is 1-5 kDa larger than their homologous counterparts from the cytosol, or from other organisms; (ii) they have a high net positive charge. Based on the modelling of three glycosomal sequences in the respective homologous structures, it is thought that the topogenic signal may consist of a unique insertion, containing one or more basic amino acids which, together with additional positive charges elsewhere, constitute two positive hot spots approximately 4 nm apart on the surface of the protein. Such common elements, unique for the glycolytic enzymes from the Trypanosomatidae, lend themselves as excellent targets for the development of new drugs.

The glycosomes of the Kinetoplastida.

Glycosomes are the microbodies of the organisms belonging to the order of the Kinetoplastida, comprising trypanosomes and leishmanias, both pathogens to man. The organelles sequester a number of glycolytic enzymes that are normally located in the cytosol in other eukaryotic organisms, and share some enzymes with peroxisomes and glyoxysomes of other protists, plants and animals. Proteins enter the glycosome by a mechanism of post-translational translocation which involves in some, but not all, cases a C-terminal oligopeptide sequence.

Expression in Bacillus subtilis of the glycosomal glyceraldehyde-phosphate dehydrogenase gene from Trypanosoma brucei.

The cloned T brucei GAPDH gene was inserted within the B subtilis GAPDH gene, carried by pUC18. Upon transformation of B subtilis by this plasmid, not able to replicate in this host, the whole plasmid was inserted in the resident chromosome, presumably by a single recombination event between homologous, chromosomal and plasmid-borne sequences. The heterologous gene was expressed, as revealed by immunological reaction with monoclonal antibodies, recognizing specifically T brucei GAPDH. T brucei GAPDH, having little or no enzyme activity, comprises about 1.56% of cellular proteins. Peptide mapping showed that a fusion of a 7.5-kDa peptide had occurred to the N-terminal part of T brucei GAPDH. This fused protein is presumably the N-terminal part of B subtilis GAPDH, in agreement with the construction of the integrative plasmid.

A rapid method for the isolation of intact glycosomes from Trypanosoma brucei by Percoll -gradient centrifugation in a vertical rotor.

A rapid method for the isolation of intact glycosomes from trypanosoma brucei is described. A post large-granule extract is subjected to density-gradient centrifugation on isotonic Percoll in a vertical rotor. This results in a good separation of the glycosomes, which equilibrated at 1.09 g/cm3, from other cellular components. Glycosomes isolated from such a gradient are 9-fold purified relative to the starting homogenate with a yield of 25%. Their glycolytic enzymes exhibit a high latency indicative of an intact glycosomal membrane.

Chemical modification of fructose bisphosphate aldolase from Trypanosoma brucei compared to aldolase from rabbit muscle and Staphylococcus aureus.

Chemical modifications of Class I aldolases from Trypanosoma brucei, rabbit muscle and Staphylococcus aureus with carboxypeptidase A, glyceraldehyde 3-phosphate and cysteine-specific reagents revealed the following differences between the three homologous enzymes. Aldolase from S. aureus was not affected by any of these reagents. Carboxypeptidase-A treatment of rabbit-muscle and T. brucei aldolase inhibited the activity of both enzymes towards fructose-1,6-bisphosphate (Fru(1,6)P2), while the activity towards fructose-1-phosphate (Fru-1-P) was affected only in the case of the trypanosomal enzyme. Moreover carboxypeptidase-A treatment reduced the turnover numbers of these two aldolases for both Fru(1,6)P2 and Fru-1-P to a similar level. Glyceraldehyde 3-phosphate, in the absence of dihydroxyacetone phosphate, also inactivated aldolases from rabbit muscle and T. brucei with second order rate constants of 1054 and 254 min-1 M-1, respectively. Using 5,5'-dithiobis-(2-nitrobenzoic acid) with rabbit-muscle aldolase, a total of 4 thiol groups could be titrated per subunit, resulting in a total inactivation. The presence of substrate completely protected the enzyme from inactivation. Methyl methanethiosulfonate also reacted with four cysteine residues, but this led to very little inactivation. This indicates that the inactivation by modification with DTNB is due to conformational changes in the enzyme. In T. brucei aldolase only one thiol group could be titrated with methyl methanesulfonate and there was no loss of activity. With 5,5'-dithiobis-(2-nitrobenzoic acid) five cysteines were titrated with an immediate and complete loss of activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of lysosomes in the uptake of macromolecular material by bloodstream forms of Trypanosoma brucei.

To investigate whether the lysosomes of Trypanosoma brucei are capable of uptake of macromolecules after internalization by the cell, we used Triton WR-1339, a non-digestible macromolecular compound, which is known to cause a marked decrease in the density of hepatic lysosomes due to massive intralysosomal storage. Intraperitoneal administration of 0.4 g/kg Triton WR-1339 to rats infected with T. brucei led to the development of a large vacuole in the trypanosomes between nucleus and kinetoplast within 22 h. Higher doses (2 g/kg) led to the disappearance of the trypanosomes from the blood and resulted in permanent cures (greater than 100 days). Lysosomes isolated from the trypanosomes of animals treated with a sub-curative dose showed a decrease in equilibrium density of 0.03 g/cm3 in sucrose gradients. These lysosomes were partly damaged as evidenced by a reduction in latency and an increase in the non-sedimentable part of lysosomal enzymes. We conclude that acid proteinase and alpha-mannosidase-containing organelles of T. brucei take up exogenous macromolecules and must therefore be considered as true lysosomes and that Triton WR-1339 acts in T. brucei as a true lysosomotropic drug. Its trypanocidal action probably results from an interference with lysosomal function.

Purification, localisation and characterisation of glucose-6-phosphate dehydrogenase of Trypanosoma brucei.

Cell-fractionation and digitonin titration of procyclic trypomastigotes of Trypanosoma brucei, revealed that almost half of the total NADP+ -dependent glucose-6-phosphate dehydrogenase (G6PDH) activity, the first enzyme of the pentose phosphate pathway (PPP), is associated with glycosomes. The specific activity of G6PDH in the purified organelles was increased 4-fold relative to a total cell extract and showed latency. Moreover, in the absence of detergents this activity was totally resistant to the action of trypsin. The cytosolic counterpart was neither latent, nor was it resistant to trypsin. Both cytosolic and glycosomal G6PDH activities behaved identically on phenyl-, CM-, heparin-, and Affigel-blue-Sepharose columns. Both isoenzymes had a subunit Mr of 62 000 and an isoelectric point of 6.85, while kinetic studies carried out on the partially purified G6PDH from both cell compartments did not reveal any differences. The purified enzyme had an apparent Km of 138 and 5.3 microM for glucose 6-phosphate (G6P), and for NADP+, respectively, and had a specific activity of 14 micromol. (min mg of protein)(-1). We conclude that while in procyclic stages of T. brucei G6PDH activity is present in two different cell compartments, i.e. the cytosol and the glycosomes, these two activities most likely represent one and the same isoenzyme.

Some kinetic properties of pyruvate kinase from Trypanosoma brucei.

We have studied the kinetics of the allosteric interactions of pyruvate kinase from Trypanosoma brucei. The kinetics for phosphoenolpyruvate depended strongly on the nature of the bivalent metal ions. Pyruvate kinase activated by Mg2+ had the highest catalytic activity, but also the highest S0.5 for phosphoenolpyruvate, while the opposite was true for pyruvate kinase activated by Mn2+. The reaction rates of Mg(2+)-pyruvate kinase and Mn(2+)-pyruvate kinase were clearly allosteric with respect to phosphoenolpyruvate, while the kinetics with Co(2+)-pyruvate kinase were hyperbolic. However, Co(2+)-pyruvate kinase was still sensitive to heterotropic activation. Trypanosomal pyruvate kinase is unique in that the best activator was fructose 2,6-bisphosphate. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate were also strong heterotropic activators, which were much more effective than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. In the presence of the heterotropic activators, the sigmoidal kinetics with respect to phosphoenolpyruvate and the bivalent metal ions were modified as were the concentrations of phosphoenolpyruvate and the bivalent metal ions needed to attain the maximal activity. Maximal activities were not significantly changed with Mg2+ and Mn2+ as the activating metal ions. Moreover, with Co2+ and fructose 2,6-bisphosphate or ribulose 1,5-bisphosphate or 5-phosphorylribose 1-pyrophosphate, the maximal activity was significantly reduced. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate resembled fructose 2,6-bisphosphate rather than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate in their action in that the K0.5 values for the former 3 compounds increased when Mg2+ was replaced by Co2+, while the K0.5 for fructose 1,6-bisphosphate and glucose 1,6-bisphosphate increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterisation of a novel iso-propanol dehydrogenase from Phytomonas sp.

An alcohol dehydrogenase with two identical subunits and a subunit molecular mass of 40,000 was purified from Phytomonas sp. isolated from the lactiferous tubes of Euphorbia characias. Digitonin titration and subcellular fractionation suggest that the enzyme is present in the mitochondrion. It utilises as substrates, primary and secondary alcohols, is specific for NAD+ as coenzyme and is inhibited by HgCl(2). The pH optimum for the oxidation of ethanol is 9.5, and for the reverse reaction 8.5. The apparent Km values for iso-propanol and ethanol are 40 and 34 microM, respectively and for the reverse reaction, with acetone as substrate, 14 microM. The respective specific activities with iso-propanol and ethanol as substrate, as measured in crude extracts are 300 and 16 mU (milligram of protein)-1. In isoelectric focusing the enzyme showed three major bands with slightly differing isoelectric points that ranged from 6.4 to 6.8. The name, iso-propanol dehydrogenase is proposed for this enzyme.

Subcellular distribution of adenylate cyclase, cyclic-AMP phosphodiesterase, protein kinases and phosphoprotein phosphatase in Trypanosoma brucei.

The subcellular distribution of adenylate cyclase, cyclic-AMP phosphodiesterase, protein kinases and phosphoprotein phosphatase in bloodstream forms of Trypanosoma brucei was determined by isopycnic sucrose-gradient centrifugation of post-large-granule extracts. Cyclic-AMP phosphodiesterase was almost entirely soluble whereas adenylate cyclase was membrane-bound. The latter enzyme appeared to be absent from the plasma-membrane fraction but copurified with acid phosphatase and acid phosphodiesterase indicating a possible association with the flagellar pocket. At least two protein kinase activities could be distinguished as based on their distribution profiles in gradients, their preference for exogenously added acceptor protein and their inhibition and stimulation by suramin and nucleoside, respectively. Suramin-sensitive protein kinase co-purified with the plasma-membrane marker alpha-D-glucosidase and a nucleoside-stimulated protein kinase behaved as a typical cell-sap enzyme. Phosphoprotein phosphatase activity was found to be mainly soluble but a small part seemed to be associated with plasma membranes.