Trypanosome ornithine decarboxylase is stable because it lacks sequences found in the carboxyl terminus of the mouse enzyme which target the latter for intracellular degradation

Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis. Mouse ODC is rapidly degraded in mouse cells, whereas ODC within Trypanosoma brucei, a protozoan parasite infesting cattle, is stable. We have expressed cloned ODC genes of both T. brucei and mouse in ODC-deficient Chinese hamster ovary (CHO) cells. The T. brucei enzyme is stable, whereas the mouse ODC similarly expressed in CHO cells is unstable. This shows that the observed difference in intracellular stability is a property of the ODC protein itself, rather than the cellular environment in which it is expressed. A chimeric ODC composed of the amino terminus of trypanosome and the carboxyl terminus of mouse ODC is rapidly degraded in CHO cells, suggesting that peptide sequences in the mouse ODC carboxyl terminus determine its stability.     

Cloning and sequencing of the ornithine decarboxylase gene from Trypanosoma brucei. Implications for enzyme turnover and selective difluoromethylornithine inhibition

Ornithine decarboxylase of the African trypanosome Trypanosoma brucei brucei had an estimated native molecular weight of 100,000 by gel filtration and a subunit molecular weight of 45,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene encoding this enzyme, present in a single copy in T. brucei, was identified by mouse ornithine decarboxylase cDNA under relatively stringent conditions of hybridization and subcloned in a 5.9-kilobase (kb) SstI fragment from a cosmid clone into the plasmid pUC 19. This clone encompassed a 2.8-kb SstII fragment that contained the entire T. brucei ornithine decarboxylase gene. The 2.8-kb SstII fragment hybridized to a 2.4-kb mRNA that presumably encodes the parasite enzyme. The 2.8-kb SstII fragment was partially sequenced and found to contain an open reading frame of 445 amino acids that has 61.5% homology with the corresponding sequence of the mouse enzyme. The only major discrepancies between the two enzymes are the addition of a 20-amino acid N-terminal peptide and the deletion of a 36-amino acid C-terminal peptide and the T. brucei ornithine decarboxylase. The C terminus has been postulated to be one of the structural factors associated with rapid in vivo turnover of mammalian ornithine decarboxylase. The absence of this C-terminal peptide in T. brucei ornithine decarboxylase predicts a slow turnover for the parasite enzyme in vivo, and this is supported by our experimental data. The lack of turnover of ornithine decarboxylase in trypanosomes may constitute the basis of selective antitrypanosomal action of the irreversible enzyme inhibitor DL-alpha-difluoromethylornithine.     

Trypanosoma brucei brucei: regulation of ornithine decarboxylase in procyclic forms and trypomastigotes

Ornithine decarboxylase (ODC) activity was measured in procyclic forms of Trypanosoma brucei brucei grown in semidefined medium. ODC activity rapidly increased in late log-phase cells which were resuspended in fresh medium. A biphasic induction curve similar to that observed in mammalian cells was observed over an 18-hr period. ODC activity increased 4.5- to 25-fold over control levels measured at zero time. Actinomycin D and cycloheximide inhibited induction by greater than 90%. Polyamines at a level not inhibitory to growth (10 microM) inhibited ODC induction, but only by 30-50%, late in the induction period. Putrescine inhibited the first peak of induction and suppressed activity at 14 hr by 75%. Polyamine analogs such as bis(ethyl)spermidine were not effective suppressors of ODC activity. The half-life of ODC in procyclic forms grown in the presence of cycloheximide was greater than 6 hr, while that of bloodstream trypomastigotes in mice treated with cycloheximide was 5 hr. A single dose of the ODC inhibitor DL-alpha-difluoromethylornithine given to infected rats or mice suppressed trypanosome ODC activity greater than 90% for more than 7 hr. These studies indicate that although trypanosome ODC increases rapidly under log growth conditions, it is less susceptible to fluctuation and external control than the enzyme from mammalian sources. The latter may be a factor in the clinical efficacy of ODC inhibitors.     

Isolation and characterization of complex I, rotenone-sensitive NADH: ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei.

Additional characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone-sensitive NADH:ubiquinone oxidoreductase of procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl-beta-D-maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second-dimensional Tricine-SDS/PAGE including the 51 kDa, PSST and TYKY subunits.     

Turnover of trypanosomal ornithine decarboxylases

Interestingly, there is a major difference in turnover rate between ornithine decarboxylases (ODCs) from various trypanosomatids. ODCs from Trypanosoma brucei and Leishmania donovani are both stable proteins, whereas ODC from Crithidia fasciculata is a metabolically unstable protein in the parasite. C. fasciculata ODC is also rapidly degraded in mammalian systems, whereas the closely related L. donovani ODC is not. The degradation of C. fasciculata ODC in the mammalian systems is shown to be dependent on a functional 26 S proteasome. However, in contrast to the degradation of mammalian ODC, the degradation of C. fasciculata ODC does not involve antizyme. Instead, it appears the degradation of C. fasciculata ODC may be associated with poly-ubiquitination of the enzyme.     

Trypanosoma brucei brucei and Trypanosoma brucei gambiense: stage specific differences in wheat germ agglutinin binding and in endoglycosidase H sensitivity of glycoprotein oligosaccharides

Gel electrophoresis, lectin affinity blotting, and endoglycosidase H digestion have been used to analyze the glycoprotein profiles of bloodstream and procyclic forms of Trypanosoma brucei brucei and T. b. gambiense. Proteins resolved by polyacrylamide gel electrophoresis were stained with silver nitrate or electrophoretically transferred to nitrocellulose and probed with a horseradish peroxidase conjugate of either concanavalin A or wheat germ agglutinin. Silver staining showed, as expected, that the expression of the variant specific glycoprotein was restricted to the bloodstream forms. Twenty-three concanavalin A binding proteins were resolved in blots of bloodstream forms. Concanavalin A binding molecules corresponding in electrophoretic mobility to 21 of these 23 bloodstream form glycoproteins were detected in blots of procyclic forms. The two concanavalin A binding glycoproteins present only in bloodstream form extracts were variant specific glycoprotein and an 81-kDa protein designated glycoprotein 81b. One concanavalin A binding molecule of 84 kDa, glycoprotein 84p, was detected only in procyclic forms. The 19 major wheat germ agglutinin binding glycoproteins expressed by bloodstream forms were not detected in procyclic forms; only small proteins or protein fragments in procyclic form extracts bound wheat germ agglutinin. Incubating transferred proteins in endoglycosidase H eliminated subsequent binding of concanavalin A to most of the 22 common glycoproteins of bloodstream forms. Three major concanavalin A binding glycoproteins of bloodstream forms, variant specific glycoprotein, glycoprotein 81b, and a 110-kDa molecule (glycoprotein 110b), and other minor glycoproteins carried sugar chains that resisted endoglycosidase H digestion. In contrast, concanavalin A did not bind to any procyclic form glycoproteins, including a 110-kDa concanavalin A binding molecule (glycoprotein 110p) after endoglycosidase H treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Haemosiderin-like properties of free-radical-modified ferritin.

Evidence was sought that the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced mouse epidermal ornithine decarboxylase (ODC, EC 4.1.1.17) activity involves both increased ODC mRNA and ODC protein. Application of 10 nmol of TPA to mouse skin led to a dramatic increase in soluble epidermal ODC activity which paralleled an increase in amount of enzymically active ODC protein as determined by gel electrophoresis of immunoprecipitated difluoromethyl[3H]ornithine-bound ODC. Application of TPA to mouse skin also resulted in an increase in ODC mRNA measured by dot-blot analysis using a radiolabelled cDNA probe. ODC mRNA induction preceded the increase in ODC activity by TPA. TPA-increased ODC mRNA displayed a single major band of 2.1 kilobases in size identified by the Northern blotting procedure.     

In vivo import of firefly luciferase into the glycosomes of Trypanosoma brucei and mutational analysis of the C-terminal targeting signal.

The compartmentalization of glycolytic enzymes into specialized organelles, the glycosomes, allows the bloodstream form of Trypanosoma brucei to rely solely on glycolysis for its energy production. The biogenesis of glycosomes in these parasites has been studied intensively as a potential target for chemotherapy. We have adapted the recently developed methods for stable transformation of T. brucei to the in vivo analysis of glycosomal protein import. Firefly luciferase, a peroxisomal protein in the lantern of the insect, was expressed in stable transformants of the procyclic form of T. brucei, where it was found to accumulate inside the glycosomes. Mutational analysis of the peroxisomal targeting signal serine-lysine-leucine (SKL) located at the C-terminus of luciferase showed that replacement of the serine residue (Serine548) with a small neutral amino acid (A, C, G, H, N, P, T) still resulted in an import efficiency of 50-100% of the wild-type luciferase. Lysine549 could be substituted with an amino acid capable of hydrogen bonding (H, M, N, Q, R, S), whereas the C-terminal leucine550 could be replaced with a subset of hydrophobic amino acids (I, M, Y). Thus, a peroxisome-like C-terminal SKL-dependent targeting mechanism may function in T. brucei to import luciferase into the glycosomes. However, a few significant differences exist between the glycosomal targeting signals identified here and the tripeptide sequences that direct proteins to mammalian or yeast peroxisomes.     

Stage-specific translational efficiency and protein stability regulate the developmental expression of p37, an RNA binding protein from Trypanosoma brucei

We have previously characterized two novel RNA binding proteins, p34 and p37, from Trypanosoma brucei. Their sequences do not show significant homology to other proteins but are highly homologous to one another. The p34 and p37 proteins are developmentally regulated, with p34 the predominant protein in the procyclic stage and p37 nearly exclusively expressed in the bloodstream cells. In vivo metabolic labeling of procyclic cells showed that p34 and p37 were differentially translated, with levels of p34 approximately fourfold higher than p37. The newly synthesized p34 and p37 exhibited differential stability in the procyclic stage. In vitro analysis confirmed this observation and further suggested that this differential stability may be due to a trypsin-like cysteine protease activity in procyclic extracts that selectively degraded the p37 protein. Taken together, these results indicate that the developmental regulation of the T. brucei RNA binding protein, p37, occurs at both translational and post-translational levels.     

Succinate secreted by Trypanosoma brucei is produced by a novel and unique glycosomal enzyme,NADH-dependent fumarate reductase

In all trypanosomatids, including Trypanosoma brucei, glycolysis takes place in peroxisome-like organelles called glycosomes. These are closed compartments wherein the energy and redox (NAD(+)/NADH) balances need to be maintained. We have characterized a T. brucei gene called FRDg encoding a protein 35% identical to Saccharomyces cerevisiae fumarate reductases. Microsequencing of FRDg purified from glycosome preparations, immunofluorescence, and Western blot analyses clearly identified this enzyme as a glycosomal protein that is only expressed in the procyclic form of T. brucei but is present in all the other trypanosomatids studied, i.e. Trypanosoma congolense, Crithidia fasciculata and Leishmania amazonensis. The specific inactivation of FRDg gene expression by RNA interference showed that FRDg is responsible for the NADH-dependent fumarate reductase activity detected in glycosomal fractions and that at least 60% of the succinate secreted by the T. brucei procyclic form (in the presence of d-glucose as the sole carbon source) is produced in the glycosome by FRDg. We conclude that FRDg plays a key role in the energy metabolism by participating in the maintenance of the glycosomal NAD(+)/NADH balance. We have also detected a significant pyruvate kinase activity in the cytosol of the T. brucei procyclic cells that was not observed previously. Consequently, we propose a revised model of glucose metabolism in procyclic trypanosomes that may also be valid for all other trypanosomatids except the T. brucei bloodstream form. Interestingly, H. Gest has hypothesized previously (Gest, H. (1980) FEMS Microbiol. Lett. 7, 73-77) that a soluble NADH-dependent fumarate reductase has been present in primitive organisms and evolved into the present day fumarate reductases, which are quinol-dependent. FRDg may have the characteristics of such an ancestral enzyme and is the only NADH-dependent fumarate reductase characterized to date.     

α-Synuclein in central nervous system and from erythrocytes, mammalian cells, and Escherichia coli exists predominantly as disordered monomer

Since the discovery and isolation of α-synuclein (α-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that α-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in α-helical structure (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107-110; Wang, W., Perovic, I., Chittuluru, J., Kaganovich, A., Nguyen, L. T. T., Liao, J., Auclair, J. R., Johnson, D., Landeru, A., Simorellis, A. K., Ju, S., Cookson, M. R., Asturias, F. J., Agar, J. N., Webb, B. N., Kang, C., Ringe, D., Petsko, G. A., Pochapsky, T. C., and Hoang, Q. Q. (2011) Proc. Natl. Acad. Sci. 108, 17797-17802). However, it remains unknown whether or not this putative tetramer is the main physiological form of α-syn in the brain. In this study, we investigated the oligomeric state of α-syn in mouse, rat, and human brains. To assess the conformational and oligomeric state of native α-syn in complex mixtures, we generated α-syn standards of known quaternary structure and conformational properties and compared the behavior of endogenously expressed α-syn to these standards using native and denaturing gel electrophoresis techniques, size-exclusion chromatography, and an oligomer-specific ELISA. Our findings demonstrate that both human and rodent α-syn expressed in the central nervous system exist predominantly as an unfolded monomer. Similar results were observed when human α-syn was expressed in mouse and rat brains as well as mammalian cell lines (HEK293, HeLa, and SH-SY5Y). Furthermore, we show that α-syn expressed in E. coli and purified under denaturing or nondenaturing conditions, whether as a free protein or as a fusion construct with GST, is monomeric and adopts a disordered conformation after GST removal. These results do not rule out the possibility that α-syn becomes structured upon interaction with other proteins and/or biological membranes.     

Characterization of glycosomal RING finger proteins of trypanosomatids

The glycosomes of trypanosomatids are essential organelles that are evolutionarily related to peroxisomes of other eukaryotes. The peroxisomal RING proteins-PEX2, PEX10 and PEX12-comprise a network of integral membrane proteins that function in the matrix protein import cycle. Here, we describe PEX10 and PEX12 in Trypanosoma brucei, Leishmania major, and Trypanosoma cruzi. We expressed GFP fusions of each T. brucei coding region in procyclic form T. brucei, where they localized to glycosomes and behaved as integral membrane proteins. Despite the weak transmembrane predictions for TbPEX12, protease protection assays demonstrated that both the N and C termini are cytosolic, similar to mammalian PEX12. GFP fusions of T. cruzi PEX10 and L. major PEX12 also localized to glycosomes in T. brucei indicating that glycosomal membrane protein targeting is conserved across trypanosomatids.     

Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli.

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.     

Trypanosoma brucei FLA1 is required for flagellum attachment and cytokinesis

The single flagellum of the protozoan parasite Trypanosoma brucei is attached along the length of the cell body by a complex structure that requires the FLA1 protein. We show here that inhibition of FLA1 expression by RNA interference in procyclic trypanosomes causes flagellar detachment and prevents cytokinesis. Despite being unable to divide, these cells undergo mitosis and develop a multinucleated phenotype. The Trypanosoma cruzi FLA1 homolog, GP72, is unable to complement either the flagellar detachment or cytokinesis defects in procyclic T. brucei that have been depleted of FLA1 by RNA interference. Instead, GP72 itself caused flagellar detachment when expressed in T. brucei. In contrast to T. brucei cells depleted of FLA1, procyclic T. brucei expressing GP72 continued to divide despite having detached flagella, demonstrating that flagellar attachment is not absolutely necessary for cytokinesis. We have also identified a FLA1-related gene (FLA2) whose sequence is similar but not identical to FLA1. Inhibition of FLA1 and FLA2 expression in bloodstream T. brucei caused flagellar detachment and blocked cytokinesis but did not inhibit mitosis. These experiments demonstrate that the FLA proteins are essential and suggest that in procyclic T. brucei, the FLA1 protein has separable functions in flagellar attachment and cytokinesis.     

Developmental variation of glycosylphosphatidylinositol membrane anchors in Trypanosoma brucei. Identification of a candidate biosynthetic precursor of the glycosylphosphatidylinositol anchor of the major procyclic stage surface glycoprotein.

The major surface antigen of the mammalian bloodstream form of Trypanosoma brucei, the variant surface glycoprotein (VSG), is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. The VSG anchor is susceptible to phosphatidylinositol-specific phospholipase C (PI-PLC). Candidate precursor glycolipids, P2 and P3, which are PI-PLC-sensitive and -resistant respectively, have been characterized in the bloodstream stage. In the insect midgut stage, the major surface glycoprotein, procyclic acidic repetitive glycoprotein, is also GPI-anchored but is resistant to PI-PLC. To determine how the structure of the GPI anchor is altered at different life stages, we characterized candidate GPI molecules in procyclic T. brucei. The structure of a major procyclic GPI, PP1, is ethanolamine-PO4-Man alpha 1-2Man alpha 1-6 Man alpha 1-GlcN-acylinositol, linked to lysophosphatidic acid. The inositol can be labeled with [3H]palmitic acid, and the glyceride with [3H]stearic acid. We have also found that all detectable ethanolamine-containing GPIs from procyclic cells contain acylinositol and are resistant to cleavage by PI-PLC. This suggests that the procyclic acidic repetitive glycoprotein GPI anchor structure differs from that of the VSG by virtue of the structures of the GPIs available for transfer.     

Determinants of the differential antizyme-binding affinity of ornithine decarboxylase

Ornithine decarboxylase (ODC) is a ubiquitous enzyme that is conserved in all species from bacteria to humans. Mammalian ODC is degraded by the proteasome in a ubiquitin-independent manner by direct binding to the antizyme (AZ). In contrast, Trypanosoma brucei ODC has a low binding affinity toward AZ. In this study, we identified key amino acid residues that govern the differential AZ binding affinity of human and Trypanosoma brucei ODC. Multiple sequence alignments of the ODC putative AZ-binding site highlights several key amino acid residues that are different between the human and Trypanosoma brucei ODC protein sequences, including residue 119, 124,125, 129, 136, 137 and 140 (the numbers is for human ODC). We generated a septuple human ODC mutant protein where these seven bases were mutated to match the Trypanosoma brucei ODC protein sequence. The septuple mutant protein was much less sensitive to AZ inhibition compared to the WT protein, suggesting that these amino acid residues play a role in human ODC-AZ binding. Additional experiments with sextuple mutants suggest that residue 137 plays a direct role in AZ binding, and residues 119 and 140 play secondary roles in AZ binding. The dissociation constants were also calculated to quantify the affinity of the ODC-AZ binding interaction. The K(d) value for the wild type ODC protein-AZ heterodimer ([ODC_WT]-AZ) is approximately 0.22 μM, while the K(d) value for the septuple mutant-AZ heterodimer ([ODC_7M]-AZ) is approximately 12.4 μM. The greater than 50-fold increase in [ODC_7M]-AZ binding affinity shows that the ODC-7M enzyme has a much lower binding affinity toward AZ. For the mutant proteins ODC_7M(-Q119H) and ODC_7M(-V137D), the K(d) was 1.4 and 1.2 μM, respectively. These affinities are 6-fold higher than the WT_ODC K(d), which suggests that residues 119 and 137 play a role in AZ binding.     

Thiol-dependent proteases of African trypanosomes. Analysis by electrophoresis in sodium dodecyl sulphate/polyacrylamide gels co-polymerized with fibrinogen

The proteases of several species of African trypanosomes were analysed by electrophoresis in sodium dodecyl sulphate/polyacrylamide gels containing fibrinogen or collagen. After electrophoresis the gels were incubated in the presence of enzyme activators and/or inhibitors and then stained with Coomassie brilliant blue. The areas where the proteolytic activity had degraded the fibrinogen did not stain and so formed clear bands against a blue background. The proteases were found to have pH optima between 5 and 6, and required dithiothreitol or 2-mercaptoethanol for full expression of their activity. They were inhibited by amino acid chloromethanes, iodoacetamide, p-chloromercuribenzoate and other inhibitors of the thiol-dependent proteases, as well as by the trypanocidal drugs berenil (4,4'-diamidinodiazoaminobenzene-diacetamidoacetate) and pentamidine [1,5-di-(4-amidinophenoxy)pentane-di-(2- hydroxyethanesulphonate)]. Trypanosoma evansi, Trypanosoma brucei brucei and Trypanosoma brucei gambiense each have a protease with a relative molecular mass, Mr, of 28 000. In addition they occasionally exhibit activity at higher Mr values (up to 105000). Trypanosoma congolense has a low-Mr protease (31 000) and may exhibit higher-Mr proteases (up to 150000). The protease profiles of Trypanosoma vivax differ from the other species, T. brucei or T. congolense, and are present in lesser amounts. The proteases of the cultured procyclic forms are present in much smaller amounts than those of the metacyclic or mammalian blood stream forms of these parasites. The catalytic properties and inhibition characteristics of these thiol-dependent enzymes suggest that they resemble the mammalian lysosomal cathepsins B and L.     

Kinetic characterization, structure modelling studies and crystallization of Trypanosoma brucei enolase.

In this article, we report the results of an analysis of the glycolytic enzyme enolase (2-phospho-d-glycerate hydrolase) of Trypanosoma brucei. Enolase activity was detected in both bloodstream-form and procyclic insect-stage trypanosomes, although a 4.5-fold lower specific activity was found in the cultured procyclic homogenate. Subcellular localization analysis showed that the enzyme is only present in the cytosol. The T. brucei enolase was expressed in Escherichia coli and purified to homogeneity. The kinetic properties of the bacterially expressed enzyme showed strong similarity to those values found for the natural T. brucei enolase present in a cytosolic cell fraction, indicating a proper folding of the enzyme in E. coli. The kinetic properties of T. brucei enolase were also studied in comparison with enolase from rabbit muscle and Saccharomyces cerevisiae. Functionally, similarities were found to exist between the three enzymes: the Michaelis constant (Km) and KA values for the substrates and Mg2+ are very similar. Differences in pH optima for activity, inhibition by excess Mg2+ and susceptibilities to monovalent ions showed that the T. brucei enolase behaves more like the yeast enzyme. Alignment of the amino acid sequences of T. brucei enolase and other eukaryotic and prokaryotic enolases showed that most residues involved in the binding of its ligands are well conserved. Structure modelling of the T. brucei enzyme using the available S. cerevisiae structures as templates indicated that there are some atypical residues (one Lys and two Cys) close to the T. brucei active site. As these residues are absent from the human host enolase and are therefore potentially interesting for drug design, we initiated attempts to determine the three-dimensional structure. T. brucei enolase crystals diffracting at 2.3 A resolution were obtained and will permit us to pursue the determination of structure.